Histology block I Flashcards
What is the first step of a glass histological slide paraffin section?
Fixation with alcohol and acetic acid of formalin for preservation of tissue degradation
What is the second step on the histological slides paraffin sections?
Embedded in paraffin or plastic for support of specimen and allows sectioning
How thick is the measure of sectioning tissues for histological slides?
~5 - 10 microns
Mention the last steps of the glass histological slides preparation
-differential staining for contrast
-dehydration through alcohol series (up to 100%)
-Mount on glass with resin and cover slip
What are the advantages of Light microscopy?
-several types [bright field, phase contrast, fluorescent, confocal]
-small and portable
-good for looking at overall arrangement
What are the disadvantages of Light microscopy?
-lower resolution, and magnification, less view.
Mention the types of electron microscopy
- Transmission electron (TEM)
-Scanning electron microscope (SEM)
What are the advantages of Electron microscopy?
-greater magnification an resolution of detail
-you can see everything
What are the disadvantages of Electron microscopy?
-Very large
-preparation of specimens and use of instrument more complex
Describe cryo-sections for light microscope
-fixed as paraffin sections
-specimen is frozen
-frozen specimen is mounted on a cryostat and sectioned with a cooled steel knife
-collected on glass slide, thawed, stained with dyes
-dehydrated and mounted under thin glass coverslip
Mention the advantages and disadvantages of cryo-sections
advantages:
-much faster and suitable while patient is on operation table
disadvantages:
-preservation of histological structure is more poor due to ice crystal damage, not best quality.
Which is the most common staining technique for light microscopy?
H&E (hematoxylin and Eosin)
identify the parts in which the Hematoxylin stains are and the Eosin stains are
Which charge is Hematoxylin and which charged strcutures does it stain?
positive; negative
Which charge is Eosin and which charged strcutures does it stain?
negative; positive
Another name for Hematoxylin staining
Basophilic-staining
What type of molecules does basophilic staining include?
Phosphate backbones, nuclei, nucleic acids and polyanions
What type of molecules does eosinophilic staining include?
collagen, cytoplasm
Another name for esosinophilic staining
acidophilic staining
how does Trichome stains work?
incorporate 3 or more dyes to give a more complex staining pattern often differentiating tissues or fibrous extracellular components from each other
Provide an example of trichome stains
Mallory staining technique
Which stain is this?
trichome stain(Mallory staining technique)
Which dyes does trichome stain use and what does it stain?
- Aniline blue
- acid fuchsin
- orange G
selectively stain collagen, cell cytoplasm, and red blood cells these colors.
mention this type of stain
light microscopy: Silver/chromium stain
what does chromium or silver stains stain?
used to stain some extracellular fibers and some cells in the nervous system and endocrine system
Mention this type of stain
Periodic Acid Schiff staining (PAS technique)
what does Periodic Acid Schiff staining (PAS technique) stain?
Periodic acid cleaves some of the carbon-carbon bonds in sugars and changes to aldehyde groups which bind the Schiff reagent and stain bright pink. Thus good for some carbohydrates and basement membranes
Mention this stains
Examples: Orcein’s elastic stain (brown); Weigert’s elastic stain (blue); Wright and Giemsa
What does Orcein’s elastic stain ; Weigert’s elastic stain; Wright and Giemsa stain do stain?
differentiate different types of blood cells
Explain step by step how are specimens for transmission electron microscopy, sectioned?
Specimen fixed with glutaraldehyde (crosslinks proteins by aldehyde groups) followed by Osmium metal (fixes lipids)
2.Dehydration of specimen with alcohol or acetone series
3.Infiltration and embedding of specimen with a polymerizable plastic
4.Cutting of section with a microtome with either a glass or diamond knife. The sections are much thinner than for light microscopy (0.1 micron)
5.Mounted on 3 mm diameter copper grid and stained with electron dense heavy metals (uranyl acetate and lead citrate)
6.Examined in electron microscope
Which kind of microscope is this?
TEM
mention the techniques for specimen fixation used for TEM?
- freeze-fracture
- Quick-freeze deep-etch technique
Explain step by step how are specimens for freeze-fracture on TEM, sectioned?
- Specimen infiltrated with a cryo-protective agent such as glycerol to help prevent ice crystal formation and then rapidly frozen by plunging into liquid nitrogen
- Frozen specimen mounted in a freeze-fracture unit at a temperature of -110 to -120 degrees under vacuum and cleaved (fractured) with steel knife
- A metal (platinum) replica of fracture face surface is made
- The specimen is removed and warmed to room temperature and the tissue dissolved away
- The cleaned metal replica of surface is examined in the transmission electron microscope
What is the freeze-fracture technique useful for?
for examining membrane structure
Mention the microscope and which structure is this?
TEM; intestinal epithelium cells
Explain step by step the quick-freeze deep-etch technique
- In this procedure the sample is rapidly frozen (-10,000 degrees/sec) without the use of cryo-protective agents. At this speed of freezing water is solidified without the formation of ice crystals which would disrupt structure.
- Freeze-fracture of the specimen is performed under vacuum maintaining the specimen temperature below -110 to -120 degrees centigrade.
- The specimen temperature is then raised to -100 degrees for a short time. At this temperature the water at the surface changes to the vapor phase and is removed from the specimen, thus exposing surface detail by etching away the surrounding water.
- The specimen is rotary shadowed with platinum to form a replica which is cleaned and examined in the transmission electron microscope.
Which technique is used here?
Quick-freeze deep-etch technique
what does the Scanning electron microscopy (sem) useful for?
has the advantage of allowing a more three-dimensional imaging of cells and tissues.
Which microscope was this taken from?
SEM
Mention the type of micrograph and all the strcutures you can recognize
Electron microscope;
1. Golgi complex
2. mitochondria
3. Rough eR
4. Smooth eR
5. secretory Vesicles
What do membranes do?
Membranes compartmentalize biochemical functions of the cell and form closed compartments within which biochemical reactions take place with minimal interference from reactions in adjacent compartments of the cell.
mention type of microscope, structure and function of organelles present
Electron microscope; mitocondria (ATP production); rough endoplasmic reticulum (protein synthesis)
Are membranes static?
No
Mention the main functions of cell membranes
- Selective permeability
- structural scaffolding
3.vesicular transport - division of labor
How does selective permeability work and mention examples?
can tightly control the environment such as pH or substrate concentrations within an organelle or region of the cell and thus influence the equilibrium or nature of the biochemical reactions taking place in that organelle; electron transport reaction, H+ act to drive prodcution of ATP, [protons]
for what does structural scaffolding work on cell membranes and provide one example?
for enzyme and receptor attachment; hormone receptors
how does vesicular transport work on cell membranes?
Forming vesicles to move large particles between cellular compartments
What is the division of labor?
In eukaryotic, the concept that cells use membranes to divide their functions into specific compartments for better efficiency
Explain this image
Fluid Mosaic Model of Membrane Structure; lipids arranged as a bilayer and proteins floating within the bilayer. Carbohydrate groups (green) are attached to both the lipids and the proteins primarily on the extracellular surface (non-cytoplasmic side) of the membrane
How are lipids arranged in the FLMMS
primarily phospholipids with some sphingomyelin and glycolipids) are arranged as a continuous bilayer with their polar head groups on surfaces and fatty acid chains in interior of bilayer
How are proteins arranged in the FLMMS?
Proteins float in the lipid bilayer like icebergs. Are present as peripheral proteins loosely attached at surface and integral proteins deeply embedded in bilayer
How are carbohydrates arranged in the FLMMS?
Carbohydrate (2 – 5% by weight) is attached on non-cytoplasmic surface to lipids and proteins
Describe de bilayer structure
is fluid like a thick oil with many (~50%) of both lipids and proteins able to move in plane of membrane as a mosaic like arrangement.
mention the components in this picture
Cholesterol molecule, hydrophobic fatty acid chain, hydrophilic polar (charged) head, non-polar non charged hydrophobic.
how are lipid and protein molecules arranged?
arranged to maximize contact of their polar groups (hydrophilic - water loving groups) with water, and to sequester their non- polar groups (hydrophobic - water hating groups) away from water (amphipathic structure)
Hows does the temperature affect the fluidity of the membrane?
higher temperature = more fluidity
lower temp = less fluidity
Hows does cholesterol affect the fluidity of the membrane?
reduces fluidity
Which components play key role in the fluidity of the membrane?
phospholipid composition, fatty acid chain length, saturation or desaturation of the fatty acid chains, temperature, and cholesterol
How does fatty acid chains affect fluidity?
Double bond promotes fluidity
Mention the components
glycophospholipids, lipid raft, carbohydrates, peripheral protein, peripheral proteins, integral protein
what does the lipid bilayer function as?
a permeability barrier
How do simple molecules pass the lipid bilayer?
simple difussion
Mention some molecules that can pass thru de lipid bilayer by simple difussion?
some gases such as O2 and CO2, small polar molecules such as H2O Etanol, small lipid-soluble molecules and some hydrophobic molecules such as benzene
how to charged molecules pass the lipid bilayer?
require integral proteins in the form of channels or carrier proteins to pass across the membrane either by facilitated diffusion (no ATP or additional energy required) or active transport (ATP or other energy expenditure required).
mention some molecules that need to pass the lipid bilayer by facilitated difucssion or active transport?
Charged molecules such as most ions, sugars, amino acids, and nucleotides
We know that charged molecules pass the lipid bilayer requiring some extra help, but how to they do this, through which structure?
Channels
How are the channels of the lipid bilayer?
can be ungated, but often are either voltage- or ligand-gated to open or close
mention the types of difussion image
Simple difussion; carrier protein; channel protein; the presence of both carrier proteins and channel proteins
to facilitate movement of large polar molecules across the membrane
What are the classes of membrane proteins?
- Peripheral proteins
- Integral proteins
Explain peripheral proteins
are loosely bound to surface of membrane by electrostatic bonds.
can you remove peripheral proteins? yes? no? how?
yes, Easily removed with high ionic strength solutions
Peripheral proteins function
Includes some cytoskeletal proteins that anchor to the membrane and are involved in shape determination and membrane stability
Explain integral proteins
are tightly bound to the lipid bilayer
Can integral proteins be removed? yes? no? how?
require membrane destruction (lipid solvents or detergents) for removal.
Mention the subdivision of integral proteins
- Transmembrane proteins
- Lipid anchored proteins
Explain transmembrane proteins
the amino acid sequences at the membrane surfaces are hydrophilic and those in bilayer are hydrophobic.
Explain lipid anchored proteins
covalently linked to lipids but do not extend thru bilayer core
mention the structures in terms of proteins
integral membrane proteins
peripheral membrane proteins
lipid anchored proteins
What is the main function of integral membrane proteins?
major proteins which act as, ion or carrier protein pumps, ion channels, receptors and signal transducing proteins (such as the G-proteins), structural linkers between cytoskeleton and extracellular matrix, enzymes, and structural proteins
Provide examples of integral proteins
describe the illustration
Diagram illustrating the important role played by integral membrane proteins which can act as channels, pumps,
receptors, linker-proteins, enzymes, and structural proteins
Where are carbohydrates located?
Mainly present on the external surface of the plasma membrane bound to some of the integral proteins and phospholipids as the cell coat or glycocalyx
What are the functions of carbohydrates?
They often act as key recognition sequences or receptor components for:
1. Enzymes
2. Hormone binding
3. Virus binding (HIV)
4. cell recognition/diferentiation (for example differences in the carbohydrate groups present on the surface of red blood cells differentiate the A, B, AB, and O blood types)
Explain the illustration in terms of carbohydrates
extracellular surface, cytoplasmic surface; Diagram of modified fluid mosaic model of membrane showing the
extracellular location of membrane-associated carbohydrates
mention the structure pointed, the tissue where it is located and the type of microscope
EM of microvilli on intestinal epithelial cells showing the glycocalyx
or cell coat
What are lipid rafts?
Larger stable regions where some of the proteins and associated lipids are arranged
Explain lipid rafts concentrations and where are they present
contain higher concentrations of glycosphingolipids and cholesterol and are present as thicker regions of the membrane.
Which is the categorization of how lipid rafts work?
These lipid rafts tend to localize together groups of proteins or lipids involved in a common function
In what are lipid rafts involved?
They may be involved in cholesterol transport, endocytosis, and especially signal transduction.
provide specific examples of lipid rafts
signal receptor and effector proteins and enzymes may be located together to receive and convey specific signals more efficiently.
What are the two major ways to look at membranes?
- Thin sections in EM
- Freeze-fracture replicas in EM
Identify the structure and the way of looking at it
- plasma membrane and zonula occludens cell junction
- Thin section EM image of intestinal epithelial cell
Identify the structure and the way of looking at it
- region of the plasma membrane and zonula occludens
- Freeze-fracture EM image
How is the structure of membranes in thin sections in TEM?
membranes have a trilaminar structure = two dense outer layers separated by an electron translucent central layer
How is the thickness of the plasma membrane and the cytoplasmic membrane in thin sections of TEM?
7.5 - 10 nm thick for plasma membrane & 6 - 7.5 nm thick for cytoplasmic membranes
Identify the structure and its appearance
Thin-section EM showing the trilaminar appearance
of the plasma membrane on intestinal microvilli
Explain this image
Electron dense, electron translucent, electron dense; trilaminar structure of plasma
membrane,
Explain how this works
charge distribution contributes to the trilaminar
appearance of the membrane as seen in thin section
EM
Explain how freeze-fracture works on TEM?
- tissue is rapidly frozen (vitreous state – no ice crystals) in cryogen and cleaved with knife edge
- Half of the tissue is cleaved away and removed in this process
- The surface of the remaining tissue is coated with a heavy metal to form a replica of the surface
- The remaining tissue is removed by bleach and the replica examined in the TEM
what is the unique feature of freeze-fracture?
the fracture plane tends to preferentially move along and split membranes along the central plane of the bilayer between the phospholipid fatty acid chains with the fracture plane passing around the integral proteins.
Name the fracture faces membrane of freeze-fracture
the P-face and the E-face are produced
What is the P-face associated with?
associated with the inner leaflet of the membrane adjacent to the cytoplasm (protoplasm).
What is the E-face associated with?
associated with the outer leaflet of the membrane adjacent to the extracellular space outside the membrane
What are intramembraneous particles? (IMPs)
An important feature seen on the fracture faces is the present of bumps which represent the integral proteins in the membrane
Mention the faces and the technique
top: E-face
bottom: P-face
Where are IMPs located and why?
In general see more IMPs on the P-face than E-face. This probably correlates with ~50% of membrane proteins being anchored in place by attachment to cytoskeletal structures or other proteins in the cytoplasm
Why is the ability of membranes to form closed spherical vesicles so important?
The vesicles and their associated proteins play a major role in the transport of substances into and out of the cell at the plasma membrane, and between organelles in the cytoplasm
What are the 3 major types of transport by membrane vesicles?
Endocytosis
Exocytosis
Intracellular transport
What is endocytosis? (short)
Is the uptake (internalization) of material by cell from the outer membrane into the cytoplasm
What is exocytosis? (short)
Is the release of material out of the cell by secretion
What is intracellular transport? (short)
Transport & sorting of proteins or other molecules between the organelles and plasma membrane.
Identify the vesicular transports
Exocytosis and endocytosis
explain endocytosis in depth
Mechanism for bulk movement of fluids, proteins, or even whole structures such as bacteria into the cell
What are the subdivisions of endocytosis
Pinocytosis
Receptor mediated endocytosis
Phagocytosis
Explain pinocytosis (short)
bulk intake of extracellular fluid, typically not highly specific
Explain receptor mediated endocytosis (short)
highly specific uptake of required substance and usually requires specific receptors for the substance and involves a protein called clathrin
Explain phagocytosis (short)
Intake of particles larger than 250 nm in diameter such as bacteria. It can be either relatively non-specific or highly specific
Mention the vesicular transport
Pinocytosis
Mention the vesicular transport
Rceptor mediated edocytosis
Mention the vesicular transport
phagocytosis
Explain in depth pinocytosis
(“cell drinking”) is a process for the non-specific (random) uptake of extracellular fluid and material in solution into pinocytotic vesicles at the cell surface
Pinocytosis is also known as
cell drinking
Pinocytosis is subdivided into:
Micropinocytosis
Macropinocytosis
identify the vesicular transport (be specific)
Micropinocytosis
identify the vesicular transport (be specific)
Macropinocytosis
identify the structure
Em of endothelial cell showing pinocytosis including macropinocytosis
briefly describe micropinocytosis
process for uptake of water and substances into vesicles less than 150 nm diameter. Constitutive (continual) process
briefly describe macropinocytosis
uptake into vesicles greater than 200 nm diameter. Allows the maximum uptake of fluid and solutes of any of the endocytotic mechanisms
describe micropinocytosis in depth
In many cells there is a continuous (constitutive) non-specific formation of small vesicles (less than 150 nm in diameter) which pinch off from the plasma membrane and carry small amounts of fluid & proteins into the cell.
Is micropinocytosis specific or non-specific?
non-specific
What does non-specific mean?
Does not require specific receptors in the plasma membrane for the ligand and does not involve the coating-protein clathrin
Which proteins are involved in the process micropinocytosis?
involves lipid rafts with the proteins caveolin or flotillin
Identify the structure and the transport
EM of continuous capillary with micropinocytic vesicles
Which proteins are found in all non-muscle cells?
Caveolin 1 and 2
Caveolin 1 and 2 can be found in all NON muscles cells except for which?
except neurons and white blood cells
Protein specific to muscle cells
caveolin 3
What other proteins are found in the micropinocytosis process, in vesicles distinct from caveolin?
flotillin 1 and flotillin 2
How does caveolin protein work?
Caveolin forms complexes of 14 – 16 monomers that aid in causing curvature of the membrane to form the vesicle.
Is clathrin requires for micropinocytosis?
No, clathrin-independent
what is dynamin and what does it do?
Is a gtpase mechanoenzyme involved in the pinching off of the vesicles allowing vesicles to be free
Is actin required in micropinocytosis?
Rearrangement of actin microfilaments of the cytoskeleton is not required
Does micropinicytosis depend on clathrin or actin?
No, clathrin and actin - independent
Is macropinicytosis sprcific or non-specific?
Non-specific
What molecules is involved in macropinocytosis?
actin-filament rearrangements
How does actin work on macropinicytosis?
to form membrane-ruffles or folds which fold-back to trap large pockets of fluid with the formation of large vesicles greater than 200 nm diameter.
Does macropinocytosis require clathrin?
No (clathrin-independent but actin-dependent)
Why is macropinocytosis used for and why is it important?
is used by cells to endocytose large amounts of fluid and trapped solutes. And In particular, it is used by cells of the immune system such as macrophages and dendritic cells to trap antigenic material to be processed to initiate immune responses
Although macropinicytosis is non specific, what regulates it?
growth factors such as macrophage colony-stimulating factor, epidermal growth factor, and platelet-derived growth factor
What is receptor mediated endocytosis?
a highly specific process for the movement of needed substances into the cell
How does RME move needed substances into the cell?
by way of binding to specific plasma membrane receptors for that substance and the formation of structures called clathrin coated- vesicles
which other proteins are involved in the binding process of the RME?
adaptins
How do adaptins work?
bind to recognition sequences of amino acids on the cytoplasmic domain of the receptor (1), and clathrin molecules in turn bind to clathrin-binding sites on the adaptin protein (2).
Mention the transport and explain
Diagram of receptor-mediated endocytosis
How does clathrin work on the surface of the membrane
acts to provide the force for bending the membrane into the shape of first a clathrin coated pit (2) which deepens and then pinches off with the help of GTPase dynamin (3) as a clathrin coated vesicle free in the cytoplasm
Which proteins are released in RME and what happens?
Clathrin & adaptin are released (5 and 6) and the vesicle fuses with early endosomal compartment of the cell
Identify the tissue, technique and mechanism
TEM image of plasma membrane prepared by quick-freeze deep-etch technique showing forming clathrin coated pits and vesicles
Describe this process
TEM images of thin sections showing stages in formation of a clathrin coated vesicle
What is the inherited condition characterized by a decreased ability of cells, particularly liver cells, to remove cholesterol from blood from receptor-mediated endocytosis of low density lipoprotein particles (LDL particles) due to a defect in the LDL-receptor protein and what does it lead to?
Familial Hypercholesterolemia; It is characterized by extremely elevated levels of cholesterol in the blood which can lead to the development of cardiovascular diseases such as atherosclerosis, heart attacks, and valve pathologies which may be fatal. Men with this condition untreated have a ~50% probability of dying from cardiovascular problems by age 50; and women a 30% probability by age 60.
Why does Familial Hypercholesterolemia happen?
1) complete loss of the receptor protein
2) or may involve defective binding of the LDL-protein particles at the LDL protein binding site
3) or may result from a mutation in the NPxY binding site for adaptin in the cytoplasmic domain of the protein thus blocking the binding to adaptin and clathrin and internalization by receptor mediated endocytosis
What is phagocytosis?
Used for intake of particles such as bacteria which are greater than 250 nm in size
By which cells is phagocytosis used and why?
Primarily used by cells of the mononuclear phagocytic System (MPS) such as macrophages and certain white blood cells to uptake bacteria and other antigenic material
By what is phagocytosis of bacteria promoted?
by the binding of antibodies or complement fragments to the bacterial surface
By what can phagocytosis be triggered also?
Phagocytosis can also be triggered by the presence of pathogen-associated recognition patterns (PAMPs) commonly found on the surface of bacteria or other pathogens
Can phagocytosis of inert non-biological materials occur?
yes, material such as carbon, particles, glass particles, and asbestos fibers
Explain this diagram
Diagram of phagocytosis for (a ) bacteria and other antigenic material which may be facilitated by binding of antibodies or complement fragments to the surface of the particle and (b) phagocytosis of non-biological particles which does not involve the binding of antibodies or complement fragments
How does phagocytosis engulfs particles and what does it form?
by cytoplasmic pseudopod-like processes to form a vesicle containing the particle which is called a phagosome
Does phagocytosis require actin, yes, no, why? Is it actin-independent or actin-dependent?
requires rearrangement of actin, phagocytosis is said to be actin-dependent
hOW DOES THE PHAGOSOME DIGEST THE MATERIAL? ans what does it form?
The phagosome fuses with a vesicle containing lysosomal enzymes to form a phagolysosome & the particle is broken down by lysosomal enzymes.
How is phagocytosis of non-biological particles different from normal phagocytosis?
Phagocytosis of non-biological particles such as glass, carbon particles, asbestos crystals is similar but does not involve the recognition of antibodies, complement fragments, or PAMPs on their surface
What is exocytosis?
is the process involved in either the release of secretory material at the plasma membrane or for transport to the endosome
Which organelles does exocytosis involve?
This generally involves the formation of proteins in the rough endoplasmic reticulum (RER) which are transported to the Golgi complex in vesicles where the protein can be modified (glycosylation and sulfation or other modifications) and packaged into vesicles for transport either to the plasma membrane or to endosomes
Identify the tissue and transport mechanism
Light micrograph of pancreatic cells showing secretory granules in their apical cytoplasm ready for secretion by exocytosis
What are the pathways of exocytosis?
constitutive secretion
Regulated secretion
Explain consecutive secretion
the vesicles carrying molecules for secretion move continuously from the trans-Golgi membranes to the plasma membrane with no storage in cytoplasm and no signal for release required
Provide examples of consecutive secretion
collagen and immunoglobulin antibody secretion
Explain regulated secretion
the vesicles carrying the molecules for secretion move from the trans-Golgi to the apical region of the cell where they are stored until secretion is stimulated by a hormone or other factor causing local movement of calcium into cell.
Provide examples of regulated secretion
the release of most hormones and pancreatic enzyme release which require specific signals for the secretion
What organelle participates in constant recycling?
endosome and plasma membrane
Explain briefly intracellular vesicular transport between organelles
Proteins for movement are made in the rER, then go to the golgi stacks, pinch off into a vesicle into another sac. Then , into the trans golgi network, packed and move to the cell surface.
Explain the orientation of the plasma membrane protein
in terms of its cytoplasmic and non-cytoplasmic domains is established during the insertion of the protein into the membranes of the rough endoplasmic reticulum. This orientation of the protein is maintained through-out the entire process of vesicular transport of the membrane protein to the plasma membrane.
what is the mechanism for fusion of the vesicles with the target organelle membrane and how are these vesicles targeted to the appropriate membrane or organelle with which to fuse?
SNARE mechanism of membrane vesicle fusion which may also provide part of the mechanism for ensuring the vesicles fuse with the appropriate target membranes
What is the SNARE mechanism?
the major mechanism for fusion of a vesicle to a target organelle or the plasma membrane and may help targeting to the correct organelle for fusion
What does SNARE stands for?
Soluble NSF Attachment Receptor protein
What are SNARE proteins?
a small class of at least 30 - 40 proteins associated both with the vesicle (v-SNARE proteins) and the target organelle membrane (t-SNARE proteins).
How does SNARE works?
They can interact specifically with each other to facilitate docking of the vesicle to the specific target organelle or plasma membrane and catalyze fusion of the membranes
The initial steps in the targetting of an endocytotic vesicle to the endosome involves
- Formation by receptor mediated endocytosis of a plasma membrane vesicle with a v-SNARE protein (synaptobrevin or other VAMP proteins) associated with the membrane
- As the vesicle approaches the endosome, a tethering protein will bind to the vesicle and draw it close to the endosome, where Rab GTPase on the vesicle and its receptor on the endosome membrane and the v-SNARE protein on the vesicle and t-SNARE proteins (such as SNAP-25 and syntaxin) on the endosome membrane will specifically interact to form a trans-SNARE docking complex.
- This docking complex will draw the vesicle and endosome membrane close enough to each other to initiate and catalyze fusion of the membranes at a fusion pore. Once fusion is initiated the SNARE complex will be referred to as a cis-SNARE complex and allow the recruitment of the general fusion proteins NSF and alpha SNAP to the docking complex to disassembly the cis-SNARE complex
Explain this illustration
SNARE mechanism. Diagram of steps in vesicle formation, targeting, docking, and fusion of a transport vesicle with the early endodome
What is Synaptobrevin/VAMP1 and what does it do?
is a V-SNARE protein in the synaptic vesicle membrane which binds with the T-SNARE proteins syntaxin and SNAP-25 to form the zipper-like SNARE complex to target and bind the synaptic vesicle to the pre-synaptic terminal membrane
What other things occur in the process of release of the synaptic vesicles containing neurotransmitters at the synapse
Fusion of the vesicle and release of its contents occurs
What does NSF (N-ethylmaleimide sensitive fusion protein) do?
functions to unzip (disassociate the cis SNARE complex) allowing recycling of the vesicle
Explain Botulinum and tetanus toxin effects at synapse
1.
The botulinum toxin from Clostridium botulinum is endocytosed into the cytoplasm of the presynaptic neuron at the synapse at the neuromuscular junction where it cleaves synaptobrevin (VAMP) a V-SNARE protein and the T-SNARE proteins Syntaxin and SNAP-25
2.
This blocks the fusion of the synaptic vesicle with the pre-terminal synaptic membrane thus blocking transmission at the neuronal synapse
3.
Tetanus toxin from Clostridium tetani is similar, but is transported to the spinal cord where it translocates to the pre-synaptic terminal of inhibitory neurons where it blocks their ability to inhibit and regulate the activity of the large motor neurons which innervate the skeletal muscles thus leading to the sustained contraction of the skeletal muscles found in tetanus
Identify the structures
Eucrhomatin, heterochromatin, nucleolus, nuclear envelope
What is chromatin? and provide an example
nuclear material
containing DNA and various
nuclear proteins (e.g. histones).
What is the nucleolus?
Site of rRNA
synthesis and contains regulat ory
cell cycle proteins
What is th enuclear envelope?
two
membrane system perforated by
nuclear pores.
What is the nucleoplasm?
nuclear content
other than chromatin nucleolus.
Identify the type of cell
Nerve cell top, bottom inactive cell (lymphocyte)
What is euchromatin?
Loosely arranged (dispersed) chromatin so that
DNA can be read and transcribed.
is eucrhomatine transcriptionallt active or inactive site of non-ribosolam RNAs (mRNA and tRNA)
Active
What is heterochromatin?
Highly condensed chromatin.
Transcriptionally
inactive.
What is consecutive heterochromatin and where is it found?
highly repetitive sequences of DNA, packaged in the same regions in different cells. Usually near the centromeres and telomeres.
What is facultative heterochromatin and where is it found?
: is not repetitive,
location is different for different cell types. It may
undergo active transcription in certain cells, under
specific conditions.
chromatin at the periphery of the nucleus
Marginal Chromatin
What are karyosomes?
Discrete bodies of chromatin irregular in size and shape and size that are found throughout the nucleus.
Chromatin found in association with the nucleolus.
Nucleolar
associated chromatin
How are nucleosomes described?
Beads on a string
Identify the structure
Chromatin isolated from interphase
nucleus appears in EM as a thread
30nm thick.
Identify the structure
Chromatin that has been experimentally
unpacked and show the nucleosomes
“beads on a
What are nucleosomes?
are the smallest units of chromatin and represent the first level of chromatin folding.
How long is the length of dna?
about 100,000 times longer than the
nuclear diameter.
How are nucleosomes packed?
8 histone molecules (octamer)
what are histones?
the principal structural proteins of chromosomes. They form a core structure of 2 units of each of the four types of
histones (H1 H4).
What is the genome?
Entire length of DNA (2.5 billion base pairs) packed in all chromosomes. Each region of
What is a gene?
Each region of the DNA helix that produces a
functional RNA molecule
What is a telomere?
The end of each chromosome is the telomere which shortens with each cell division
Whats the role of telomerase?
Telomere length plays an important role in the lifespan of cells.
Identify
neutrophil from a female blood smear, Barr body
What is a barr body?
discovered in nerve cells of female cats, looks like a drum stick, can tell the sex
mention some aneuploidies
Trisomy
21 (Down’s): leads to
mental retardation and congenital
malformations.
Abnormalities
in sex chromosomes
Kleinfelter’s
Syndrome (XXY):
Male phenotype and no
secondary sex characteristics.
Turner’s
Syndrome ( XO):
What is cytogenetic testing?
Special fluorescence probes
(FISH technique ) for in situ hybridization
are used. A single fluorescent DNA or a mixture of different probes is
used to produce different colors in each chromosome.
Deescribe
Chromatin condensation; protamines replace histones in tightly compacted chromatin
Describe
Chromatin condensation; protamines replace histones in tightly compacted chromatin
Which proteins houses the nucleolus and what do they do?
nucleolin and nucleostemin ,
which are shuttling proteins that relocalize from the nucleolus to the nucleoplasm.
What is nucleostemin?
Nucleostemin is a p53 (tumor supressing protein) binding protein found within the nucleolus that regulates the cell cycle and influences cell differentiation
Identify the strcuture
Fibrillar centers, fibrillar material, granular material
What are fibrillar centers?
contain DNA loops
of five different chromosomes (13, 14,
15, 21, and 22) that contain rRNA
genes, RNA polymerase I, and
transcription factors.
What is fibrillar material?
(pars fibrosa) contains ribosomal genes that are actively undergoing transcription and
large amounts of rRNA.
What is granular material?
(pars granulosa): represents the site of initial
ribosomal assembly and contains
densely packed preribosomal particles.
What is the nucleonoma?
the network formed by the granular and fibrillar materials .Genes for the ribosomal subunits are localized in the interstices of this network.
What is the nuclear envelope?
Membrane barrier that separates
the nucleoplasm from cytoplasm
(encloses chromatin)
By what is the nuckear envelope separated?
Separated by a perinuclear cisternal space , which is continuous with cisternal space
of the rough endoplasmic reticulum
What perforates the nuclear envelope?
nuclear pores , which mediate the active
transport of proteins between nucleus and cytoplasm.
What is th emain fucntion of th enuclear lamina?
Support the nucleus
Identify
Electron micrograph of a portion of the nuclear lamina from a Xenopus oocyte. It is formed by intermediate filaments (lamins) that are arranged in a square lattice. The nuclear lamina is a meshwork of
intermediate type filaments.
What is the nuclear lamina formed by?
intermediate filaments lamins and lamin
associated proteins. Mainly composed of Lamin A nad lamin C
Which protein attaches the fibrous (nuclear lamina) and which are the lamin receptors it interacts with?
orthogonal lattice. Attached via lamin B
protein to the inner nuclear membrane
through its interactions with lamin receptors:
nurim (binds lamin);
emerin (binds lamin A and B;
lamin B receptor (binds lamin B)
Identify the strcuture
Fibrous lamina (nuclear lamina)
When do lamins dissemble?
during mitosis
functions of the nuclear lamina:
The nuclear lamina appears to serve as
scaffolding for chromatin, chromatin
associated proteins, nuclear pores, and the
membranes of the nuclear envelope.
*
Essential for many nuclear activities
including DNA replication and transcription,
it is involved in nuclear organization, cell
cycle regulation, differentiation, and gene
expression. Lamins disassemble during
What are laminopathies?
Mutations of lamin and their associated (receptor) proteins cause various diseases called laminopathies. These are tissue specific diseases that affect striated muscle, nerve or skeletal development, and premature aging.
provide examples of laminopathies
Emery Dreifuss muscular dystrophy EDMD
Hutchinson Gilford Progeria Syndrome (HGPS),
Explain Emery Dreifuss muscular dystrophy EDMD
associated with mutations in either lamins or lamin receptors, characterized by muscle
weakness and wasting in the upper and lower limbs and cardiomyopathy (weakening of heart muscle). The X linked recessive form of EDMD caused by mutations of emerin , whereas autosomal dominant form of
EDMD caused by mutations in lamin A/C proteins.
Explain Hutchinson Gilford Progeria
Syndrome (HGPS),
One of the most severe laminopathies, Hutchinson Gilford Progeria Syndrome (HGPS), which is caused by aberrant splicing of the gene encoding for A type lamins and is associated with premature aging.
Identify the strcutures
nuclear pores
What are nuclear pores?
Array of “openings” (70
to 80 nm) through the nuclear envelope
formed from the merging of the
inner and outer membranes.
What is the nuclear pore complex and what does it do? (NPC)
composed of multidomain proteins
collectively referred to as
nucleoporins. Regulate the
passage of proteins
between the nucleus and
cytoplasm.
*
Ribosomal
subunits exported from
nucleolus to the cytoplasm.
Nuclear
proteins imported from
cytoplasm to nucleus.
Identify the structure
Nuclear Pore Complex with Freeze Fracture
Identify the strcuture
Nuclear Pore Complex Negative Staining Techniques
What does the NPC composed of?
NPC is composed of a central cylindrical body placed between inner and outer octagonal rings, each consisting of 8 protein particles.
What is Cryoelectron Tomography (CET)
CET is a version of
electronmicroscopy where 3D reconstruction ofa sample (i.e., electron tomogram) is created from 2D images obtained from freeze fracture methods.
identify the micgrography
Cryoelectron Tomography (CET)
Identify the strcutures
The Nuclear Pore Complex
Explain Import NPC, does it require energy?
Large molecules
containing a nuclear localization amino acid sequence
(NLS , Pro Lys Lys Lys Arg Lys Val), can be imported into the nucleus.
*
Labeled NLS
proteins destined to nucleus binds to cytosolic nuclear importin
receptors that directs them to the NPC in a GTP dependent fashion controlled
by Ran Ras like nuclear GTPase signaling.
Explain Export NPC
Proteins that
posses the nuclear
export sequence (NES) bind in the
nucleus to exportin and to a Ran
GTP molecule.
*
The
exportin Ran GTP complexes
pass through the NPC into cytoplasm
where GTP is hydrolyzed and the
NES protein is released.
*
The
NPC transports proteins, all
forms of RNA, as well as ribosomal
subunits in their fully folded
configurations.
Identify mechanism?
Export (Nuclear Pore
Complex)
*
Proteins that
posses the nuclear
export sequence (NES) bind in the
nucleus to exportin and to a Ran
GTP molecule.
*
The
exportin Ran GTP complexes
pass through the NPC into cytoplasm
where GTP is hydrolyzed and the
NES protein is released.
*
The
NPC transports proteins, all
forms
Identify mechanism
Import (Nuclear Pore
Complex)
What is the nucleoplasm?
Material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus.
Explain this nuclear alteration
Nuclear alterations in dying cells
Pyknosis
: condensation of chromatin leading to shrinkage of the
nuclei (dense basophilic masses).
*
Karyorrhexis
: is the destructive fragmentation of the nucleus of
a dying cell whereby its chromatin is distributed irregularly
throughout the cytoplasm
*
Karyolysis
: disappearance of nuclei proteins due to complete
dissolution of DNA (increased DNAase).
What is necrosis?
Exposure to physical or chemical
environment that causes acute cellular injury and damage to the plasma membrane
What initiates necrosis?
This damage may also be initiated by viruses, or proteins called perforins
Mention characteristics of necrosis
Rapid cell swelling and lysis are two characteristic features of this process
When does apoptosis occur?
Occurs during normal embryologic development or physiologic
processes, such as follicular atresia in the
ovaries Cells
How does apoptosis occur?
Cells can initiate their own death through
activation of an internally encoded suicide
program Apoptosis
Mention characteristics of apoptosis
Apoptosis is characterized by controlled
autodigestion, which maintains cell membrane integrity thus, the cell dies without spilling its contents and damaging its neighbors
Identify cell death
necrosis left apoptosis right
identify
This electron micrograph shows an early stage of apoptosis in a
lymphocyte. The nucleus is already fragmented, and the irreversible
process of DNA fragmentation is turned on. Note the regions containing
condensed heterochromatin adjacent to the nuclear envelope
b.
Further fragmentation of DNA. The heterochromatin in one of the
nuclear fragments (left) begins to bud outward through the envelope,
initiating a new round of nuclear fragmentation. Note the reorganization
of the cytoplasm and budding of the cytoplasm to produce apoptotic
bodies.
c.
Apoptotic bodies containing fragments of the nucleus, organelles, and
cytoplasm. These bodies will eventually be phagocytosed by cells from
the mononuclear phagocytic system. (Courtesy of Dr. Scott H.
Kaufmann.)
Identify
This photomicrograph taken with light microscopy of intestinal
epithelium from the human colon shows apoptotic bodies (AB) within a
single layer of absorptive cells.
Identify structure and protein stain
Breast cancer cell
Orange: Microtubule marker
Purple: Actin microfilament marker marker
What is the cytoskeleton?
The cytoskeleton is a network of protein filaments and tubules in the cytoplasm composed of 3 types of non-membranous filamentous organelles
Mention the non-membranous filamentous organelles that compose the cytoskeleon
microfilaments
intermediate filaments
microtubules
What is the diameter of the microfilaments and its protein?
6-8 nm diameter & actin is the main protein
What is the diameter of the intermediate filaments and its proteins?
are 10 nm diameter & are composed of intermediate filament proteins (several classes of proteins
What is the diameter of the microtubules and its proteins?
24 nm diameter & tubulin (α and β
tubulin) is the main protein
What is one of the major key roles of the cytoplasm?
cell shape determination and motility
Identify the structure, type of micrograph and protein
Fluorescently light micrograph with the distribution of intermediate filaments in fetal lung fibroblasts cells in culture/ Red=vimentin (intermediate filaments)
green=actin
Identify the structure, type of micrograph and protein
Fluorescent staining of microtubules with (green) and nucleus in blue. gren= immunostaining for microtubules (tubulin)
What is cytomotor function of the cytoskeleton?
movement: of whole cell, organelles, cilia, chromosomes (spindle formation) (mt), and the processes of cytokinesis (mf), endocytosis, exocytosis, cytoplasmic streaming [release of vesicles]
What is cytosekeletal function of the cytoskeleton?
involvement in cell shape determination; shape of cilia and microvilli
Identify strcuture, tissue, cell shape
Micrograph of non-distended transitional bladder epithelium. Note top balloon-shaped (umbrella or dome) cells or columnar/cuboidal-like shape cells in the bottom
Identify strcuture, tissue and protein
EM micrograph of intestinal microvilli supported by actin microfilaments.
Identify structure, tissue and region
Tracheal epithelium showing cilia on apical
surface supported by microtubules forms these motile cilia
WHat does tubulin have in common in both cytoskeletal functions?
tubulin in cilia helps maintain the long linear shape of the cilium but also plays a key role in the movement of the cilia
Identify strcuture, function
tubulin maintains the shape of the colium in this oviduct and helps with the moevment of ciclia to move the oocyte along the oviduct
Identify strcuture, function
Micrograph of cerebellum showing the very asymmetrical shaped Purkinje cells with dendrites
Identify strcuture, function
EM image of a mature myelinated axon showing microtubules in the cytoplasm
Explain correlation of cytiskeleton in purjunkie neurons of cerebellum
cytoskeleton plays a role both in maintaining very asymmetric shape of Purkinje neurons in cerebellum (structural function), but also within axons plays a key role in fast axonal transport of molecules and organelles to axon terminal (motor function)
How is actin called in its unpolymerized and polymerized form?
G-actin (Globular), while in its polymerized form (the filament) it is called F-actin (Filamentous
What is the strcuture of the filament of actin?
double helical arrangement
describe structure and stain
Electron micrograph of actin (negative stain)
describe structure and stain
computer filtered image of actin filament, and diagram of double helical structure of actin filament
Mention functions of the microfilaments
Anchorage and movement of membrane proteins = focal adhesion point
Formation of the structural core of microvilli and stereocilia
*
Locomotion of cells – thought to involve a reversible gelation - solation of cytoplasm & may involve myosin II (thick filaments) and or myosin I
*
Extension of cell processes – filopodia & lamellipodia
*
Vesicular transport and movement of organelles (myosins I, II, Va, Vb, VI, VIIa, IXa, X)
*
Cytokinesis
*
Morphogenetic movements of tissues during embryology e.g. shaping of epithelia into tubes and etc
What are focal adhesion points?
Points where you have actin filaments intercating with integrins and integrins interacting with the extracellular matrix; scaffolds for signaling pathways
Why is polymerization and depolymerization of actin microfilaments important?
There is a large pool of G-actin in the cell in equilibrium with the F-actin. This allows microfilaments to be polymerized and depolymerized in specific areas of the cell as necessary for function
What does actin polymerization require?
requires the hydrolysis of ATP
On what does actin polymerization depend on?
depends on the local concentration of G-actin (binds to ATP) & other actin-binding proteins.
Explain briefly polymerization of actin
The polymerization occurs as a polarized process in which G-actin molecules with bound ATP bind preferentially at the (+)-end of the filament. After a short time the bound ATP is hydrolyzed to bound ADP, and eventually the actin with bound ADP is lost by depolymerization at the (-) end of the filament. This is an ongoing process which has certain proteins to stop it.
Which process is this?
Polymerization of actin
filaments
What is treadmilling?
the name given to this process in which new actin molecules add preferentially to (+) end filament, while other actin molecules are lost at (-) end thus giving a continuous flow actin from (+) end to (-) end
explain the diagram
Pulse-Chase Experiment Showing Treadmilling. Labeled actin molecules (green) add at the plus end of microfilament and after a short period of time the labeled molecules in solution are replaced with non-labeled actin and the patch of labeled actins can be seen to move along the filament
Where does treadmilling play a key role?
in motility
Mention Drugs That Interfere with the Polymerization and Depolymerization of Actin Microfilaments
Cytochalasins B and D; Phalloidin
How does Cytochalasins B and D interfere with the Polymerization and Depolymerization of Actin Microfilaments
These drugs bind (+) end of microfilament & block addition of new actin molecules. Thus the drugs can block cellular activities that require polymerization of the microfilaments, such as cell movement and cytokinesis
How does Phalloidin interfere with the Polymerization and Depolymerization of Actin Microfilaments
This drug stabilizes microfilament structure & prevents depolymerization. Thus blocks cell activities requiring reversible polymerization & depolymerization such as cell movement and cytokinesis
how does cytochalasis b,d and Phalloidin interefere?
These drugs disrupt the equilibrium between G-actin and F-actin. Because drugs block mitosis by inhibiting cytokinesis they may have anti-tumor use. The drugs also inhibit such cellular activities as lymphocyte migration, phagocytosis
How is actin arranged in the cell?
As a loose meshwork of crosslinked filaments or as bundles of parallel filaments
Where is the loose meshwork of crosslinked filaments of actin found in the cell?
typically found immediately under and attached to the plasma membrane or as a loose meshwork throughout the cytoplasm.
Which crosslink proteins are often associated with te actin filaments in loose meshworks?
Filamin and spectrin
Which proteins crosslink with the bundles of parallel filaments in the actin arrangement?
fimbrin or villin
Identify the structure and the actin arrangement
Electron micrograph (EM) showing microvilli at apical surface of cell as finger-like projections with a core consisting of a bundle of actin
filaments (terminal web)
Identify the structure and the actin arrangement
A higher magnification micrograph (EM)
showing microvilli at apical surface of cell as
finger-like projections with a core consisting of
a bundle of actin filaments
Identify the structure and the actin arrangement
core bundle; loose meshwork; EM of cell prepared by quick-freeze deep-etch technique
showing microvilli with core bundle of actin filaments and actin filaments as a loose meshwork in cytoplasm below microvilli
Identify the structure and the actin arrangement
Distribution of actin filaments in pulmonary
artery cells in culture.
Green= imunostaining of actin stress fibers (bundles of crosslinked microfilaments)
red=imunostaining of mitochondria
How much % of protein does actin make?
10%
How does actin microfilament facilitate the binding of a multitude of different proteins
Actin is extremely conserved in its amino acid sequence
Mention proteins that can help the filament carry out its function
crosslink filaments into loose gel (filamin)
*
fragment filaments (gelsolin)
*
bundle filaments into bundles (villin, fimbrin, espin)
*
interact with actin to produce motion including vesicular transport (myosins I, II, Va, Vb, VI, VIIa, IXa, X)
*
cap filament ends (example gelsolin) [prevent filament from linking polymerization or shortening length by depolymerization]
*
attach filaments to cell membrane (spectrin, myosin I, ezrin)
*
sequester actin monomers to prevent filament formation (profilin & thymosin)
Which myosin is found in the muscle and what does it do?
myosin II; involved in muscle contraction; however there are approximately 35 of unconventional myosins invovled in vesicular transport and movement of other cargo such as organelles
Which myosins are associated with vesicular transport and endosomal vesicles?
myosins V and VI
Why is myosin VI of extreme interest?
it moves in a retrograde direction towards the (-) end of the actin filament unlike all other known myosins. Thus bidirectional transport by myosins on actin filaments is possible
What does tubulin do in these movement of vesicles and other cargo by unconventional myosins V and VI
transports long distances
How is the movement along the actin-filaments in areas that lack microtubules and provide example?
often short-range motion, such as under the plasma membrane
What is the measurement of microtubules and its key role?
24 nm in diameter. Play a role in both cytoskeletal and cytomotor functions
Identify structure
EM micrograph of microtubules in the mitotic spindle of a dividing cell.
What are MAPs of MW 50,000 – 300,000 daltons?
microtubule associated proteins involved in many functions
Mention the functions of cytoplasmic microtubules?
Intracellular vesicular transport and organelle movement– for example as in fast axonal transport
*
Maintenance of cell shape
*
Movement of cilia and flagella
*
Cell elongation and movement (cell migration)
*
Chromosome movement (spindle formation and pulling chromosomes to poles)
*
Maintenance of position of ER and intermediate filaments (if disrupt microtubules, then get a shift in position of both endoplasmic reticulum and intermediate filaments)
What are microtubules composed of?
Composed of tubulin - dimers arranged in 13 -15 protofilaments.
By what are cytoplasmic microtubules disrupted?
cold, pressure, or high levels of calcium
Does actin containing microfilaments have polarity, if so where?
yes, in the cytoplasmic microtubules
Where do the tubulin alpha-beta dimers align?
align in a head to tail conformation along the protofilaments. Thus the two ends of the microtubule are different
Is polymerization of microtubule polar or non polar? why?
polar with tubulin dimers with bound GTP preferentially binding at the plus end of the microtubule and loss of the tubulin dimers being promoted when the tubulin has bound GDP.
Which proteins add to the stability and blocking lengthening & shortening of cytoplasmic microtubules?
Capping proteins and other bound proteins
what is the equilibrium of cytoplasmic microtubules?
dynamic equilibriuk (dynamic stability) between assembly and disassembly.
what correlates to tje rapid shortening and lengthening of the microtubule?
the state of the nucleotide bound to the tubulin dimer with bound GTP = assembly at plus end and bound GDP = dissociation)
Mention drugs that interfere with microtubules
Colchicine, Taxol, Vinblastin & vincristine
How does Colchicine interfere with microtubules? and what is it used for?
binds tubulin dimers & blocks their addition to tubule end causing rapid depolymerization of the microtubules. Is used for treatment of some cancers and gout arthritic symptoms. For gout it acts by blocking the movement and function of immune cells in the areas of inflammation in the joints.
How does Taxol interfere with microtubules? and what is it used for?
binds to tubulin in the intact microtubule & stabilizes the microtubule. Is used in therapy for breast cancer (very toxic)
How does vinblastine & vincristine interfere with microtubules? and what is it used for?
bind microtubule dimers and increase their binding to each resulting in the formation of paracrystalline deposits of the dimers thus reducing the ability of microtubules to polymerize. Are used in the treatment of leukemia and various types of lymphomas in particular. (stabilizes too much, if you stabilize too much or destabilize it affects either way)
How do drugs that interfere with microtubules treat cancer?
used as anti-mitotic and anti-proliferative drugs in cancer therapy because they can block mitotic activity through their action on the polymerization and depolymerization of microtubules (can be highly toxic)
Explain the polarity of microtubules
There is polarity in the arrangement of the ends of the microtubule in the cell.
With what are the (+) and (-) ends associated regarding microtubule polarity and what are the exceptions?
In most cells the (-) end of the tubule is associated with the mitotic organizing center (centrioles) near nucleus & microtubules grow & radiate out with (+) end toward cell periphery. The exception is that in dendrites of neurons they may have opposite orientation. In axons these have a typical orientation.
In microtubules of nervous tissue (dendrites) where is the (+) located?
near the nucleus
identify the strcuture and the polarity
Immunolabeled microtubules growing outwards from mitotic organizing center (centrioles) with (-) end at center and (+) end peripherally
why is polarity so important in microtubules?
important in determining how cytomotor proteins bind microtubules and produce movement.
Which proteins move towards the (+) end and which move towards the (-) end?
(kinesins) move toward the (+) end
& others (cytoplasmic dyneins) toward the (-) end
given the polarity of microtubules, they are said to give which type of movement to organelles, vesicles and molecules?
bidirectional movement
Mention How many types and classes of kinesis and dyneins are there?
kinesins (40 types in 14 classes) and dyneins (9 heavy chain classes)
What do kinesins and dyenins represent and what are they invovled in?
represent families of proteins with many distinct members and are involved in a variety of movements including fast axonal transport, chromosome movement during mitosis and meiosis, movement of cilia and flagella.
Identify strucutres
Immunostaining showing the presence of a kinesin (Eg5 – red staining) which interacts with polar spindle fibers from the two mitotic poles to help separate the chromosomes. Dynein (not stained) also acts by binding at kinetochores and drawing chromosomes towards poles; chromosomes are shown in the middle
What is an example of retrograde transport virus?
Rabiesvirus is transmitted through direct contact (such as through broken skin or mucous membranes in the eyes, nose, or mouth) with saliva or brain/nervous system tissue from an infected animal. People usually getrabiesfrom the bite of a rabid animal.
Herpes simplex virus may use retrograde transport
How is rabies virus mechanism retrograde mechanism and its symptoms?
Evidence has been shown that the rabies virus is transmitted along neuronal axons from near the site of the bite to the brain by retrograde transport involving binding of the P protein from rabies virus to the dynein light chain protein LC8.
In the brain, rabies causes acute inflammation of the brain leading to extremely aggressive and violent behavior and death if not treated
What is the diameter of intermediate filaments?
10-12 nm
wHAT IS THE MAIN FUNCTION OF INTERMEDIATE FILAMENTS?
provide a cytoskeletal (structural, support). They provide mechanical strength and control cytoplasmic stiffness in individual cells, provide mechanical strength to layers of cells, help to control cell shape, and may play a role in the positioning of organelles in the cell
Identify
EM showing
hemidesmosomes at the base of
an epidermal cell. The filaments are
Keratin-containing Intermediate
filaments
Identify
EM showing a
desmosomal junction (intercellular)
with intermediate filaments (arrows [zonula occludens]
Mention the first 2 classes of intermediate filaments proteins
Keratins (epithelia) –tonofilaments
mention class 3 intermediate filaments proteins
Vimentin & vimentin-like proteins including desmin (muscle) & glial fibrillary acidic protein (glial cells in nervous system) & several less well known proteins
Mention class 4 intermediate filaments proteins
Neurofilament protein (neurons)
mention class 5 intermediate filament proteins
Lamins (nucleus - all eucaryotic cells)
Mention class 6 intermediate filament proteins
Beaded filaments – (eye lens fiber cells)
What is a way of determining origin of cancers and tumors?
Antibody labeling of the different classes of intermediate filament proteins (particularly the different subclasses of cytokeratins
Identify clinical correlation and strcuture
For example this image shows astrocytes, a type of glial cell (supporting cell) in the brain immunostained for glial fibrillary acidic protein (GFAP) in their intermediate filaments.
A tumor in which the intermediate filaments of the cells are immuno-stained with GFAP would suggest the tumor is of glial cell origin.
Similarly, immunostaining of the intermediate filaments with an antibody to keratins would suggest an epithelial origin.
Section of brain immuno-stained for glial fibrillary acidic protein
(GFAP) showing astrocytes (a type of glial supporting cell) stained because
their intermediate filaments contain GFAP
How are intermediate filaments assembled?
assembled from a pair of helical monomers that twist around each other to form coiled-coil dimers. Intermediate filaments are assembled in a dense ring around the nucleus and then radiate out toward the cell periphery.
Do intermediate filaments have polarity? yes? no? how does it affect?
No polarity, it is difficult for a protein to walk into a direction due to no polarity no (+) end
How does the non-polarized unit of the intermediate filament come along?
Intermediate filaments are assembled from a pair of helical monomers that twist around each other to form coiled-coil dimers. Two of these dimers then assemble with each other in antiparallel fashion (parallel but pointing in opposite directions) to generate a staggered tetramer of two coiled-coil dimers, thus forming the non-polarized unit of the intermediate filaments. Each tetramer then binds to other tetramers staggered relative to each other along the axis of the filament to form a protofilament, 8 of which are crosslinked to form the intermediate filament
How are intermediate filaments assembled in some epithelial cells?
they attach at cell junctions such as hemidesmo-somes and desmosomes and extend as bundles across the cytoplasm from junction to junction
Which are the least understood of the cytoskeletal filaments in function?
Intermediate filaments
Are intermediate filaments stable or dynamic structures, why?
Although previously thought to be stable structures, they are now considered to be dynamic structures which form filamentous linkages between the cell surface and the nucleus that change depending upon the mechanical stresses on the cell.
What are iNTERMEDIATE FILAMENTS important regulating what?
important regulators of the intracellular changes in cytoplasmic mechanics that accompany various physiological activities such as cell contraction, migration, proliferation, and organelle positioning
What do intermediate filaments provide?
They provide stiffness and strength to individual cells and help the cells resist and respond to environmental stresses. maintain location of organelles within the cell
What do intermediate filaments provide in epithelia and how?
help to strengthen an entire layer of cells. Thus providing a mechanism in which tension or pulling forces on the cells are shared by all the cells thus providing strength to the layer.
What are lamins in intermediate filaments
How IF provide strength to the nuclear envelope
are intermediate filaments involved directly in motility? why or why not?
No; due to their lack of a polar structure which hinders cytomotor proteins binding to them and moving along the filaments in only one direction.
Although intermediate filaments are not directly involved with motility, what are they involved that is important additionally?
rearranged during cell motility & may be involved in controlling directionality of the movement of the cell, and may play a role in the formation and directionality of cytoplasmic processes in cells such as astrocytes.
Explain the The Epithelial-Mesenchymal Transition (EMT) clinical consideration and its significance
During embryological movement epithelial cells undergo a process called the epithelial-mesenchymal transition in which the epithelial cells undergo a change to a more mesenchymal-like cell morphology. A key feature of this process is that the intermediate filament composition changes from cytokeratin-containing intermediate filaments to vimentin-containing intermediate filaments and the breakdown of desmosomal and hemidesmosomal junctions. The cells then assume a more mesenchymal – fibroblast-like morphology and become motile.
The clinical significance of this is the finding that epithelial cells becoming cancerous typically recapitulate this epithelial-mesenchymal transition to become motile and metastatic. Evidence has been shown for example that vimentin expression is required for the invasive behavior and metastasis of both prostate and breast cancer cells
Mention the proteins which bind intermediate filaments
Filaggrin
Plectin family of proteins
Plakins
Lamin-Associated Proteins
what does Filaggrin do in IF?
binds keratin filaments into bundles
What do plectin family of proteins do in IF?
bind microfilaments, microtubules, and intermediate filaments into 3-D networks
What do plakins do in IF?
involved in binding of intermediate filaments to hemidesmosomes and desmosomes. They also bind actin filaments to the neurofilaments in sensory neurons. Includes desmoplakins, desmoplakin-like proteins, and plakoglobulins
What doe lamin-associated proteins do in IF?
Emerin, lamin B receptor, nurim, and other lamin-associated polypeptides [serve to form the fibers coding on the inner surface of nuclear envelope]
Identify
Schematic of 3D structure of the G-actin with ribbon representation
What is filopodia?
slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating
What is lamellipodia?
cytoskeletal protein actin projections that occur at the leading edge of the migratory cells.
Identify
filopodia, lamellopodia
What is apical and what is basal?
apical is to the outside and basal is close to the base or connective tissue.
What is the endoplasmic reticulum?
Membrane bound closed system of
anastomosing tubules and broad flat
saccules called cisternae
With what is the ER continuous with?
the outer membrane of the nuclear envelope
mention the main functions of the smooth endoplasmic reticulum (sER) and where is it welld eveloped and which enzyme related to it?
principal organelle involved in detoxification and conjugation of noxious substances. It is particularly well developed in the liver and
contains a variety of detoxifying enzymes related to cytochrome P450 into
sER plasma membranes.
Whih compouds does the sER detoxify and how?
They modify and detoxify hydrophobic compounds such as pesticides,
barbiturates and carcinogens converting them into water-soluble conjugated
products that can be eliminated from the body
Mention other functions of the sER
-lipid and steroid metabolism,
-glycogen metabolism, and calcium homeostasis.
-membrane formation and recycling
Mention other enzymes associated with the sER
hydrolases, methylases, glucose-6-phosphatase, ATPases
Idenfity structure
The ER Network
identify structure
Heat shock protein 90 B-1 antibody (green):
HSP90B1, ER protein resident required for
the folding of numerous signaling proteins
What is the sER?
Consists of short anastomosing tubules that are not associated withribosomes.
What do large amounts of sER contribute to?
cytoplasmic eosinophilia (acidophilia)
when viewed in the light microscope.
Identify strcutures
Abundant smooth ER in a steroid-hormone-secreting cell. This electron micrograph is of a
testosterone-secreting Leydig cell in the human testis. Can see the cisternae
identify structures and function
Electron Microscopy (EM) image of abundant sER usually present in cells with active lipid synthesis. detoxyfication
How is the sER called in the muscle?
sarcoplasmic reticulum a specialized ER
Whats the function of the sarcoplasmic reticulum?
Its function is essential in the process of muscle contraction. In basal state (no contraction) it sequesters Ca+2 from the
cytoplasm. for movement.
Where is the sarcoplasmic reticulum located?
The sarcoplasmic reticulum is close to the plasma membrane invaginations that conduct the activating impulses, which trigger the release of Ca+2 and produce the contraction
Identify the sarcoplasmic reticulum
blue web
identify structure and function
Rough endoplasmic reticulum (rER). A region of the endoplasmic reticulum that is associated with ribosomes and is thus the organelle in which the synthesis of proteins takes place
(proteins destined for export, to a specific organelle or to the plasma membrane)
What staining does the rER has and why?
The RNA in the ribosomes has basophilic staining. Therefore, the rER has a basophilic staining pattern.
Identify structure and satining
Pancreatic acinar cells; lumen and basophilic staining rER; the clear part is acidophilic staining
What is the portion of the cytoplasm that has basophilic staining, called?
ergastoplasm
identify structure and staining
Light micrograph of nerve cells (H&E) with Nissl bodies. Nerve cells have large basophilic bodies aclled nissl bodues (rER + many free ribosomes)
identify structure
Electromyograph of nerve cells showing abundant rER
identify structures
chief cell = secretory cell. Electron micrograph of rER in a chief cell of the stomach
shows membranous cisternae “C” closely packed in “parallel arrays”. The particles attached to the exterior surface of the
membrane are ribosomes
Short description of ribosomes?
the protein making machine
What are ribosomes and where are they made?
Small particles 15 to 20 nm in diameter and contain ribosomal RNA and protein (made in nucleoulus)
What is the ribosomal small subunit?
(40S): 1 RNA molecule + 33 proteins
What is the ribosomal large subunit?
(60S) binds to membrane of rER: 3 RNA molecules + 49 proteins
How can a ribosome participate in protein synthesis?
associating with mRNA
What are polyribosomes?
polysomes = clusters of 10-20 ribosomes
What do polyribosomes in the cytoplasm do?
synthetize proteins that remain in the cytoplasm.
Identify structure
spiral shaped polyribosomes. Electron micrograph of the rough endoplasmic reticulum (rER) and polyribosome complexes
Identify structure
The large spiral cytoplasmic assemblies (arrows) are chains of ribosomes that form polyribosomes that are actively engaged in translation of the mRNA molecule.
The first 15 to 60 amino acids on the
amino-terminus forms a ________
signal sequence (signal peptide SP)
The signal peptide interacts with a _________
signal recognition particle (SRP)
Explain the cascade of protoein synthesis for export snd sorting sequence
The first 15 to 60 amino acids on the
amino-terminus forms a signal sequence
(signal peptide SP)
* The signal peptide interacts with a signal
recognition particle (SRP)
* The complex SRP–polyribosome arrests
polypeptide synthesis and moves to the
rER membrane
* SRP binds to a docking protein of rER
* Now, synthesis resumes with peptide
going into lumen of rER.
* Dissociation of the SRP–docking protein
complex from the ribosome and rER
membrane occurs
* Protein synthesis continues towards the
lumen of the rER
Identify structureS
Electron micrograph of the rER and polyribosome complexes. We can see pores, nucleous, nuclear membrane, cisternae of rER
Mention some modification that occur by enzymes in the proteins synthesized into the lumen of the rER and why?
-Glycosylation
– Disulfide-bond
– Hydrogen-bond
– Protein folding is achieved with the help of proteins called chaperones.
they need more processing
Why is the rER a quality checkpoint?
Properly modified proteins are concentrated in a lumen of the rER to be delivered to the Golgi. Proteins secreted by the constitutive pathway, are delivered to the Golgi apparatus within minutes
Explain the alpha-1 antitrypsin deficiency
a communication problem between rER and Golgi Apparatus. characterized by inability of rER to export a mutated protein to
Golgi apparatus.
In alpha1- antitrypsin (A1AT [gets accumulated in the rER without doing its function]) deficiency, one single amino acid substitution causes
accumulation of defective A1AT within the rER cisterna of liver hepatocytes. The
result is impaired liver function. Also the lack of enzyme contributes to cause
emphysema in the lungs. (basically damages the alveolos of lung)
Identify structure and explain
Electron micrograph of a hepatocyte from the liver of a child with α1-antitrypsin deficiency. The arrow shows the accumulation of
α1-antitrypsin within the rER causing impaired function
Identify structure and what does it relate to
Emphysema; alpha1- antitrypsin (A1AT) deficiency
How to proteins move from rE to the golgi?
in small transport vesicles
Identify structure
Electromyograph of hepatocyte showing rER and Golgi apparatus. also transport vesicles full of proteins to be transported to the golgi
Who’s the FedEx of the cell?
the golgi apparatus
Mention the two classes pf vesicles involved in the transport of proteins
COP I nad COP II
Explain COP I
mediates transport vesicles originating in the Golgi back to rER, as well as retrograde transport between Golgi cisternae. This
retrograde transport returns proteins mistakenly transferred to the Golgi
Explain COP II
coats the transport vesicles responsible for the anterograde transport that mediates transport of proteins from rER to Golgi.
Identify
Camillo Golgi’s drawings of cells in spinal ganglia (1898).
What was the “internal reticular apparatus?
In 1897 Camillo Golgi studying the nervous
system with his “black reaction” (silver
impregnation) described an intracellular
structure called like that
Explain the staining of the golgi by camilo
Impregnated with silver or osmium (osmiophilic) appears as juxtanuclear structure
How does the golgi look in LM?
appeared unstained with most histological stains including hematoxylin or eosin (H&E). The term “negative Golgi” was used for that unstained region of the cytoplasm
How does the golgi look in LM?
parallel stacks of flattened closely apposed, membrane-bound saccules or cisternae, and associated vesicles
Where is the golgi present?
present in all cells and it is well developed in secretory cells
Identify
PLASMA CELLS (secretory cells that produce
immunoglobulins) – “Negative Golgi”
Identify
Negative golgi, lack of staining in the apical region
Identify strcutures
Electron micrograph of Golgi Apparatus; cis-golgi network, rER; vesicles
Describe the Cis-golgi network forming face and its coposition
Convex cisterna, osmiophilic, closest to rER. Mannose phosphorylation (M-6-P) occurs here (for proteins that go to the lysosomes). It stains in EM.
Describe the intermediate compartment of the golgi and its composition
Nicotinamide adenine dinucleotide phosphatase (NADPase)
* N-acetyl glucosamine transferase (agt)
Describe the trans-golgi network maturing face and its coposition
Concave cisterna, stains for sialyl transferase and galactosyl transferase
* Thiamine pyrophosphatase activity.
* Site of condensing vacuoles and vesicles
Identify
The Golgi apparatus in a cultured fibroblast stained with a fluorescent antibody that recognizes a Golgi resident protein.
Mention the functions of the golgi
- Posttranslational modification, sorting
and packaging of secretory products
synthesized in rER. - Synthesis of some carbohydrate
components of complex glyco-proteins
associated with glycosyl transferase.
GAGs, glycoproteins, glycolipids,
proteoglycans. - Trimming of oligosaccharides in
glycolipids and glycoproteins - Transport and modification of
membrane proteins and glycolcalyx - Mannose-6-phosphate is added to
proteins destined for lysosomes (phosphorilation of proteins)
Identify structures and regions
Condensing vacuole in formation for cytoplasm, secretory cell. This electron micrograph shows the extensive Golgi apparatus in a cell of the pancreas. The
flattened membrane sacs of the Golgi apparatus are arranged in layers. The cis-Golgi network (CGN) is represented by the flattened vesicles on the outer convex surface, whereas the flattened vesicles of the inner convex region constitute the trans-Golgi network (TGN). Budding off the TGN are several
condensing vesicles (vacuoles) (1). These vesicles are released (2) and eventually become secretory
vesicles (granules)
What are sorting signals in the golgi?
attached to proteins, determine intracellular location/destine of the protein. Sorting signals can be linear polypeptide arrays or carbohydrate molecules
Mention the four major pathways of protein secretion from the Golgi disperse proteins to various cell destinations
apical plasma membrane (mas sencillo)
basolateral plasma membrane
endosomes or lysosomes
apical cytoplasm
Explain the apical plasma membrane pathway
Many extracellular proteins and membrane
proteins are delivered to this site.
The constitutive pathway most likely
uses non-clathrin-coated vesicles
Explain the basolateral plasma membrane pathway
Proteins with specific sorting signal attached to them by the TGN. This constitutive pathway uses vesicles coated with unique proteins that bind to epithelium-specific adaptor proteins. The transported membrane proteins are continuously incorporated into the basolateral
membranes. Present in most polarized epithelial cells.
Explain the endosome or lysosome pathway
The majority of proteins destined to
organelles have specific signal
sequences. Enzymes destined to
lysosomes use Mannose-6-phosphate (M-6-P) markers and are delivered to early or late
endosomes. occurs in the cis with specific signal
Explain the apical cytoplasm pathway
Proteins stored in large secretory vesicles undergo a maturation process and are retained within the vesicle. Eventually fuse
with plasma membrane to release secretory product by exocytosis. Characteristic of secretory cells in exocrine glands. they have to live in the cytoplasm for a while until the signal arrived for them to be secreted.
identify the pathways
apical plasma membrane (mas sencillo)
basolateral plasma membrane
endosomes or lysosomes
apical cytoplasm
Identify the structure
Light micrograph of pancreatic acinar cells (H&E). Apical region showing secretory granules
Identify the structure
Electron micrograph of pancreatic acinar cells showing mature secretory granules (S)
Identify the structure
Transfer Electron Microscopy (TEM) shows a system of cytoplasmic
membrane-enclosed compartments associated with all endocytotic
pathways. AN early endosome.
What is the stable compartment model?
Describes early and late endosomes as stable cellular organelles connected by vesicular transport with the cell exterior and with the
Golgi apparatus. Coated vesicles fuse only with the early endosomes because they have specific surface receptors
What is the maturation model?
Early endosomes are formed de novo from endocytotic vesicles, their
composition changes as some components are recycled between the
cell surface and the Golgi. The maturation leads to the formation of
late endosomes and then to lysosomes
What is the major function of the early endosomes?
to sort and recycle proteins internalized by endocytotic pathways
How is the pH comparison in early endosome to lysosome, why?
in early endosome is less acidic, more basic, in late endosome it turn more acidic and in lysosome is acidic in order to degrade . Each transport add a proton H+ to make the environment more acidic for degradation
What are lysosomes
Membrane-bound organelles, containing
50 or more hydrolytic enzymes. They are
responsible for the degradation of
macromolecules derived from
endocytotic pathways and the cell itself
Where are lysosomes found?
Present in all cells. Abundant cells of
inflammatory pathways (macrophages
and neutrophils)
Lysososme function
sites of intracellular digestion
What is endocytosis and autophagy?
-Endocytosis: Macromolecules
brought into the cell (cell “eating”)
– Autophagy: Turnover of cellular
components (cell “eating itself”)
How are lysosomes stained?
Acid phosphatase staining:
Histochemical method used to identify
lysosomes. However eaarly endosome doesnt stain here.
Identify staining and structure
Electron micrograph of phosphatase
acid reaction staining in lysosome.
Identify
lysosomes, mitochondria; primary lysososme since it hasnt digested what its going to degrade.
Where is M-6-P present?
In early and late endosomes, lysosomes and golgi
TO which enzyme does several enzymes attach in the lysososmal emembrane
M-6-P
Describe the membrane of the lysosomes?
resistant to the hydrolytic digestion occurring in their lumen. *Unusual phospholipid
structure: Cholesterol + lysobisphosphatidic acid
Mention the structural lysosomal membrane proteins
-Lysosome-associated
membrane proteins
(LAMPs)
* Lysosomal membrane
glycoproteins (LGPs)
* lysosomal integral
membrane proteins
(LIMPs)
* Proton H+ pumps,
transport proteins.
Explain the constitutive secretory pathway
Proteins secreted to the cell > surface > endocytosed > early endosomes > late endosomes > lysosomes.
Explain the The Golgi-derived coated vesicle
secretory pathway
Proteins exit the Golgi in clathrin-coated vesicles > late endosomes > lysosomes
How do lysosomes digest small extracellular particles?
are internalized by both receptor mediated endocytosis and pinocytosis (red arrows) -> endosome -> late endosome -> lysosome
How do lysosomes digest large extracellular particles?
such as bacteria and cellular debris are
delivered via the phagocytotic pathway (blue arrows) -> phagosome -> late endosome -> lysosome
What is autophagy?
when the cell uses lysosomes to digest itself by creating a membrane from sER
What are the types of autophagy?
-macroautophagy
-microautiphagy
-chaperone-mediated autophagy
What is macroautophagy?
a portion of the cytoplasm or an entire organelle is surrounded by an
intracellular membrane of the endoplasmic reticulum to form a double-membraned
autophagosome vacuole. After fusion with a lysosome they are degraded
What is microautophagy?
cytoplasmic proteins are internalized into lysosomes by invagination of the lysosomal membrane
What is chaperone-mediated autophagy?
to lysosomes is the most selective process for degradation of specific cytoplasmic proteins. Participation of proteins called chaperones. The chaperone protein (hsc73) binds to the protein and transports it into the lysosomal lumen, and it is degraded.
What is this structure?
Autophagosomes
What is this structure?
Undigested residues from both autophagy and heterophagy form Residual bodies. Also called lipofuscin pigment when identified in neurons with light microscopy techniques
Can cells destroy proteins without lysosomes? yes? no? how?
Yes, via proteosomes which destroy abnormal proteins misfolded, short-lived regulatory proteins, transcriptional factors.
Are proteosomes ATP dependent or independent?
ATP dependent
Which aggregations take part in proteosomes?
Ubiquitin 4 molecules
What happens if degradation with proteosomes does take place? or if there is accelerated degradation?
accumulation of tau protein (alzheimers); human papilloma virus
What is the fucntion of mitochondria?
Provide energy for biosynthetic and motor activities of cells, mir=tochondrial DNA encodes 13 enzymes involved in oxidative phosphorilation, 2 rRNAs, 22 transfer (RNAs).
What are TIM and TOM complexes?
Chaperone proteins assist in translocation of proteins
What is this structure?
Electron micrograph of skeletal muscle, showing abundant mitochondria in the
intermyofibrillar spaces in striated muscle cells
What us this structure?
Mitochondria
What is this structure?
Fluorescent microscopy of cells were fixed and stained with phalloidin stain
conjugated with fluorescein dye. Phalloidin binds and stabilizes actin filaments,
preventing their depolymerization. Accumulation of actin filaments at the
periphery of the cell just beneath the plasma membrane. These cells were also
stained with two additional dyes: a mitochondria-selective dye (i.e., MitoTracker
Red) that allows the visualization of mitochondria (red) in the middle of the cell
and DAPI stain that reacts with nuclear DNA and exhibits blue fluorescence over
the nucleus
What is this structure?
Mitochondria within hair cells of a sensory system in fish. 3-D reconstructions of
sensory hair cell patches with mitochondria labeled with Mito-Tracker in white.
Scale Bar: 5um.
describe the outer mitochondrial membrane
Permeable, voltage-dependent anion channels called porins. Posses TOM complexws for importing proteins into the intermembrane space. Also contains Acetyl CoA, phospholipase A2, monoamine oxidase, synthase
Describe the inner mitochondrial space
impermeable, has cristae for amplification of furcae area for efficiency of energy generation. Steroid metabolism, wich in phospholipid cardiolipin makes the membrane impermeable of ions.
Whta is this structure?
Elementary particles, for oxidative phosphorilation, for ATP generation.
Describe intermembrane space
Space between the inner and outer membranes and contains specific enzymes that use ATP. These enzymes include creatine kinase, adenylate kinase, and cytochrome c. The latter is an important factor in initiating apoptosis
Describe mitochondrial matrix
Surrounded by the inner mitochondrial membrane and contains the soluble enzymes of the citric acid cycle (Krebs cycle) and the enzymes involved in fatty-acid β-oxidation
Describe the matrix granules of mitochondria
Store Ca2+ and other divalent and trivalent cations. Mitochondria can accumulate cations and regulate the concentration of cations in the cell, a role they share with the sER. The matrix also contains mitochondrial DNA, ribosomes, and tRNAs
What is this structure?
matrix granules
What is this structure and mechanism?
mitochondria dividing by fission
Which is the proapoptotic protein?
BCl2 regulates eprmeability adn leads to irreversible release of cytochrome c
What is the orthodox configuration and condensed configuration?
1) The orthodox configuration, the cristae are
prominent, and the matrix compartment
occupies a large part of the total mitochondrial
volume. This configuration is observed in
healthy cells.
2) The condensed configuration is
characterized by unfolded cristae that are not
easily recognized and the intermembrane space
increases to as much as 50% of the total volume
of the organelle. These changes expose
cytochrome c and facilitate its release from the
mitochondria during apoptosis.
What is this strcuture?
orthodox config and condensed config
emention some effects of mitochondrial defects
Metabolically active tissues that use large amounts of ATP, such as muscle
cells and neurons, are the most commonly affected.
Myoclonic epilepsy with ragged red fibers (MERRF)
* Is characterized by muscle weakness, seizures, and cardiac and
respiratory failure. Microscopic examination of muscle tissue from
affected patients shows aggregates of abnormal mitochondria.
* MERRF is caused by mutation of the mitochondrial DNA gene encoding
tRNA for lysine (to add a lysine amino acid). This defect affects the
electron-transport chain of respiratory enzymes affecting ATP production
What are peroxisomes?
Small membrane bound organelles, containing more than 40 oxidative enzymes. Lack acid phosphatase (found in lysosome). H2O2 (hydrogen peroxide) is a product of oxidation reactions and it is a toxic substance. Excess hydrogen peroxide is destroyed by the enzyme catalase.
Where are peroxisomes abundant?
Abundant in liver and kidney, the number of peroxisomes increases in
response to diet, drugs, and hormonal stimulation. In liver, peroxisomes
are responsible for detoxification of ingested alcohol
What does the nucleoid of the peroxisome have?
crystalloid of urate oxidase
What is this structure?
Peroxisomes; crystalloid of urate oxidase
WHat is this structure?
Glycogen (alpha particles) in liver
What si this structure?
Electron micrograph of skeletal muscle showing mitochondria and abundant amount of glycogen particles (beta)
What is this structure?
lipid droplets, energy store abundant in sER
What is this structure?
Melanin pigment
What is this structure?
Lipofuscin pigment, brownish gold; wear and tear, cellular aging.
What is lipofuscin?
Lipofuscin is a aggregation of oxidized lipids, phospholipids, metals, and organic molecules that accumulate within the cells as a result of oxidative degradation of mitochondria and lysosomal digestion.
What is this structure?
Electron micrograph of aneuron showing a group of neurofilaments and residual
bodies (lipofuscin pigment in light microscopy
What is this strcture?
microvilli, brush border of small intestine
What is this structure?
Epididymis with stereocilia
What is this structure?
Tracheal epithelium with cilia
What is this structure?
cilia
What is this structure?
microvilli of intestinal “finger-like structures”
What is this structure?
stereocilia
What is this structure?
cilia
What is the core of microvilli? and what is the border?
bundle of parallel microfilaments, striated border
What is the main function of microvilli?
absorption, increase surface area for better absorption.
What is this structure?
periodic acid stain, stains carbohydrates due to glycocalyx in microvilli
What is this structure?
microvilli
What is this structure and its components?
microvilli on intestinal cell showing central bundle of actin-containing microfilaments
What is this structure?
microvilli with core bundle of actin filaments and actin filaments as a loose meshwork in cytoplasm below microvilli
What is this structure?
Bundle of actin filaments in cross section in microvilli
Mention the proteins in the molecular structure of microvilli
cross-linked: fimbrin, fascin, espin
attaches to plasma membrane: myosin I
anchored at tip: villin
terminal web: myosin II, spectrin, alpha-actinin
Where can we found stereocilia?
Found in male reproductive tract in the epididymis and vas deferens where they are involved in and regulate absorption of fluids from semen and in hair cells in the inner ear for perception of sound
What is the stereocilia structure?
Have actin bundle at core
crosslink proteins: espin, fimbrin and fascin can be present
villin is absent
attachement to memebrane: ezrin instead of myosin I
alpha-actinin can be cross-linked to other stem regions.
What does stereocilia do in the inner ear?
act as mechano-sensors of fluid movement in the ear which is perceived as different sounds, bending of hair cell causes calcium channels to open and depolarize the cel and stimulate adjacent auditory nerve.
What is this structure?
scanning EM of stereocilia in the inner ear. (b) Espin labeled by fluorescent
green marker can be seen adding at the tip of the stereocilia. (c) diagram showing the
molecular mechanism for the addition of new actin molecules and crosslinking
moleculesat the tip of the stereocilia
What is threadmillinf in stereocilia?
constant renewal of actin and crosslink proteins at the + tip, repair mechanism
What is usher syndrome?
Usher syndrome is characterized by hearing loss, visual impairment due to retinitis pigmentosa (RP), and in some cases, vestibular dysfunction. Usher syndrome is the most common cause of deaf-blindness and an important etiology underlying autosomal recessive deafness.
Proteins encoded by Usher genes have been found to be particularly abundant in the stereocilia and the synaptic regions of hair cells and guide the cohesion and development of the hair bundles. Mutations in these proteins typically lead to deafness thus showing the importance of the stereocilia and the maintenance of their structure,
Where can we find cilia and how they differ from microvilli and stereocilia?
on the apical surface of many epithelial cells. They differ from both microvilli and stereocilia in having a core structure composed of an arrangement of microtubules and by their ability to move (in some types)
Mention the 3 types of cilia
-Motile cilia which can move
-primary cilia (monocilia, are imotile but have sensory function
-nodal cilia (a type of monocilium that can move and its important for embryological development on te ventral node
Where can we find motile cilia?
commonly found on cells which are involved in moving material along their apical surfaces such as in the respiratory system (mucus) and oviduct (the oocyte), or in the form of flagella for moving the entire cell (sperm
What is this structure?
motile citia
What is this structure?
motile cilia in the trachea for movement of mucus.
What is this structure?
motile cilia in the oviduct (thicker)
How is the moelcular structure of cilia called?
axoneme
How is the axoneme characterized?
9+2 arrangement of microtubules
by what are the 9 doublets connectedto teh 2 central microtubules?
radial spokes
Protein that connect the microtubule doublets
nexin-linking protein
How is the arrangement of the basal body in motile cilia?
9 triplet strcuture
What dies dyenin do in motile cilia?
the bending of cilia.
How can the doublets in motile cilia be divided?
a-subfier (13 protofilamets) and b-subfiber (10-11 protofilaments)
What is this structure?
basal bodies of motile cilia
Identify structures
Nexin link, radial spoke, a-subfiber, b-subfiber, dyenin arms
What is the effcetive stroke, the recovery stroke and metachronal rythm in motile cilia?
Requires ATP hydrolysis. This is the forward effective stroke.
*
The elastic recoil of the nexin and radial arm proteins then returns the cilium to original position (recovery stroke)
*
The adjacent cilia beat in a coordinated metachronal rhythm which is a wave of movement to rpopel fluid like trapped bacteria along the epithelium
how do basal bodies arise?
either by duplication of existing centrioles (centriolar pathway) or by a denovo pathway (acentriolar pathway)
Protein that anchors basal body to cytolasm
rootletin
identify
basal bodies, axoneme, basal foot, striated rootlet, transitional zone
identify
basal bodies
What is the pattern of primary cilia (monocilia)
have a 9 + 0 pattern of microtubule doublets, no dyenin arms; non-motile
Where can we find monocilia?
Are present on most cells in epithelial, connective, muscle and nervous tissues
identify
monocilia (primary cilia)
What are some functions of primary cilia?
monitor fluid flow through the kidney tubules, biliary tubules, brain ventricles, and auditory system.
*
There presence has been found to be critical for function in the organs in which they are present
What dies polycystine-1,2 do?
initiate signaling cascades within the cell by movement of fluid.
Whst is the nodal cilia and what are they important for?
unique form of monocilium found on cells in the region of the ventral node in the developing embryo and are thought to be important for determining the left-right patterning of the embryo
What is the pattern of nodal cilia?
+ 0 pattern of microtubule doublets like primary cilia but are motile since they have dynein arms. This distinguishes them from primary cilia (monocilia), only asingle ciliu present
Identify
This figure shows a scanning EM (A) of nodal cilia on the surface of nodal cells in the ventral node
identify
Nodal cilia
Why is the rotation “like a propeller” of nodal cilia important
In the region of the primitive node in the embryo they rotate in a clock-wise manner and generate a leftward or “nodal” fluid flow which is detected by sensory receptors on the left side of the embryo and helps to establish left-right symmetry of the organs.
What can happen if there are defects in the proteins of nodal cilia?
cause “situs inversus” in which the heart and other organs appear on the wrong side of the body in approximately 50% of the patients with the defects
What proteins acts as transport motor up and motor down in Intrafagellar transport in cilia (IFT)
kinesin II; dyenin
Mention the function of IFT?
provides precursor molecules to tip of cilia and removes old molecules
what are some mutations in IFT?
Mutations in genes encoding the proteins in the IFT-A and IFT-B complexes result in loss of cilia or result in ciliary dysfunctions.
For instance, mutations in the genes for kinesin II the main anterograde transport protein in cilia (KIF3A/KIF3B/KAP3 trimeric complex) can result in either very short or absent cilia; resulting in ciliary disfunction related to a number of clinical conditions called ciliopathies.
What are some ciliopathies (primary cilia dyskinesia or immotile cilia syndrome?
*
Autosomal dominant polycystic kidney disease (ADPKD) resulting in cysts in kidney leading to renal failure, and can also cause cysts in pancreas and liver
*
Chronic infections of the lungs & upper airways due to inability of the respiratory cells to move mucus along their surface. For example, Kartegener’s syndrome.
*
Situs Inversus with inversion of the side on which organs occur. As noted above this may be primarily related to defects in the nodal cilia
*
Infertility in males due to immotile sperm
*
An increased rate of ectopic pregnancy in affected females that cannot properly move the oocyte along the oviduct
*
Hydrocephalus internus (accumulation of fluid in the brain ventricles) may occur in patients due to defective motile cilia on the ependymal cells lining the cerebrospinal fluid-filled spaces in the brain
Explain parthologies involving primary cilia ADPKD
Autosomal dominant polycystic kidney disease (ADPKD) is the major type of polycystic kidney disease (PKD). It is characterized by formation of multiple fluid-filled cysts which destroy the renal cortex leading to more than half of patients progressing to renal failure.
*
ADPKD is associated with mutations in the Pkd1 and Pkd2 genes which encode for the proteins polycystin-1 (PC1) and polycystin-2 (PC2) in the membrane of the primary cilium. These proteins normally interact to form an ion channel complex that is necessary for signaling pathways and for the mechanosensory function of the primary cilium. The exact mechanism by which the mutations lead to formation of the cysts, however, is still unclear.
*
Individuals with ADPKD often exhibit other pathologies not associated with the kidney that are attributed to ciliary abnormalities. These include cysts in the pancreas and liver that are accompanied by an enlargement and dilation of the biliary tree system
What is kartager’s syndrome?
Congenital abnormality typically involving absence of the motor protein dynein from motile cilia and nodal cilia
Kartagener’s Syndrome can result in defects in ability of respiratory epithelial cells to transport mucus (mucociliary transport) in the tracheo-bronchial tree. This results in chronic respiratory disorders (bronchitis and sinusitis), otitis media (inflammation of middle ear), persistent cough, and asthma
Identify pathology
Lack of dyenin arms, kartagener’s syndrome
