Unit 3: Cell Structure + Staining Flashcards
What are the different types of microscopes?
Light microscope, fluorescent microscope, electron microscope
What are the types of light microscopes?
Bright field microscope (regular)
Phase contrast microscope (specialized)
Dark field microscope (specialized)
How do light microscopes (bright field) work?
light source emits visible rays, the condenser focuses the rays onto a sample, the light that is absorbed by the sample produces contrasts that help reveal structural details of the specimen (cells/tissue), the objective lens captures the rays, and it is passed to the ocular lens or a video camera
What is the limit of resolution (resolving power of a microscope)?
The extent to which a microscope can distinguish fine details in the specimen as separate, distinct image points
What is used in A?
Bright field microscope
What is used in B?
Phase contrast microscope
What is used in C?
Interference-contrast optics
What is used in D?
Dark field microscopy
Why is phase contrast microscopy used?
enables us to visualize unstained, live cells
Why is dark field microscopy used?
to visualize small structures (ex. Microtubules with a diameter of 25nm)
How do phase contrast microscopes work?
shows differences in the refractive index of parts of the cells as different areas of brightness and darkness. This is due to a phase plate between the illumination source and the condenser lens
How do dark field microscopes work?
opaque disc is placed on the center of the condenser so light only passes around its edges => results in light scattering, which makes specimens appear bright against a dark background.
How can the resolving power of a microscope be increased in an electron microscope?
resolving power of a microscope is a function of the wavelength of the illuminating source and the numerical aperture of the lens system being used
the resolving power can be increased by using an illuminating radiation of a shorter wavelength
What are the types of electron microscopes?
Transmission electron microscope
Scanning electron microscope
How are electrons and wavelength related to resolving power of an electron microscope
have a shorter wave length of 0.004 nm, and are used to increase the resolving power of the microscope. The electrons are particles, as well as exhibit wave-like behavior
What are the techniques like for preparing tissues/cells for electron microscopy?
almost similar to that used for light microscopy
What is atomic force microscopy used to visualize?
one of the most powerful tools for studying the surface topography at molecular and atomic resolution
How does atomic force microscopy work?
a very sharp, pointed probe, approaching the size of a single atom at the tip, scans the specimen following parallel lines along the x-axis, repeating the scan at small intervals along the y-axis.
The sharp tip is mounted at the end of a very flexible cantilever so that the tip deflects the cantilever as it
encounters the atomic force on the surface of the specimen
Does atomic force microscopy use illumination?
This is a non-optical microscope which works in the same
way as a fingertip, which touches and feels the skin of our
body when we cannot see it.
How are transmission electron microscopes and scanning electron microscopes different?
What is immunostaining?
process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistry or IHC) or fluorescence (immunofluorescence or IF)
What is the membrane which surrounds the cell?
Plasma membrane
A typical cell membrane consists of
Lipids, proteins, and carbohydrates
What is a cell membrane?
an essential feature of every cell which defines the boundaries of the cell and delineates its various compartments
What is the structure of cell membranes?
6-10 nm wide, resembles a railroad track when observed by electron microscopy. The outer, dark lines are 2 nm thick while the central electron lucent space is 3.5 nm thick.
How has the structure of cell membranes been found?
The structure has be elucidated by the use of TEM and freeze-fracture studies as well as light microscopy of living cells and has resulted in the fluid mosaic model
What are the functions of the cell membrane?
Boundary and permeability barrier
-Organization and localization of function
-Transport process
-Signal detection
-Cell-to-cell communication
What is the fluid mosaic model? *
this is the generally accepted model.
phospholipid bilayer in which the hydrophilic heads of the molecules are directed toward the outside of the cell and the cytoplasm. The hydrophobic tails are found within the membrane away from the aqueous surfaces. Because of this amphipathic property, the lipid molecules form a bilayer in aqueous environments
What are intrinsic/integral proteins?
have hydrophilic domains and hydrophobic domains situated in the middle of the bilayer
may have enzymes linked to them, as well as glycoproteins, receptor proteins, and cholesterol
How can intrinsic proteins be visualized?
freeze-fracture replicas by TEM and they appear as bumps or depressions on the E-face and P-face. In these preparations they are called intramembranous particles (IMPs).
What are examples of intrinsic proteins?
Rhodopsin, glycophorin
What are examples of extrinsic proteins?
Aldolase, glyceraldehyde 3-phosphate dehydrogenase
Is the cell membrane fluid or static? Do intramembranous particles move?
Fluid
IMPs may have considerable movement along the phospholipid bilayer unless they are otherwise prevented by cell junctions or other modifications
What are phospholipids and structure? *
the most abundant lipids
unique structure with Amphipathic nature with hydrophobic and hydrophilic regions
What are the phospholipids found in the cytoplasmic side of the bilayer?
Phosphatidyl ethanolamine, Phosphatidyl serine, Phosphatidyl inositol
What are the phospholipids found in the extracellular side of the bilayer?
Phosphatidyl choline, Sphingomyelin
What are the other other lipid molecules in the bilayer?
Glycolipid - found on all plasma membrane surfaces 5% of the lipid in outer monolayer
What are the functions of glycolipids?
Receptors for extracellular molecules
Receptors for bacteria
Protection
What are examples of glycolipids?
Galactocerebroside, GM ganglioside and Sialic acid (Neuraminic acid)
The fluidity of the lipid bilayer depends on
Composition
What do eukaryotic plasma membranes contain which enhance the permeability of membrane?
Cholesterol
What does cholesterol do in the plasma membrane? *
Regulates the rigidity of the membrane
limits the movement of adjacent phospholipids and makes the membrane less fluid, and more mechanically stable
What does the outer part of the lipid bilayer consist of?
predominantly of phosphatidyl choline, sphingomyelin,
and glycolipids
What does the inner part of the lipid bilayer consist of?
aminophospholipids such as phosphatidyl serine, phosphatidyl ethanolamine and phosphatidyl inositol
Phosphatidyl serine contributes a negative charge in the inner membrane
Where is cholesterol in the lipid bilayer?
Both inside and outside
What is the functional importance of lipid bilayer asymmetry? *
Protein kinase C (PKC), when activated, binds to negatively charged phosphatidyl serine on the inner membrane Negatively charged environment is essential for PKC function
Phosphoinositol - inner membrane and involved in signal transduction
What is permeable and impermeable in the lipid bilayer?
Permeable: small nonpolar molecules (O2 and CO2)
Impermeable: all ions and charged molecules (no matter how small)
How much of the membrane do membrane proteins constitute?
Generally 50%
What are the functions of membrane carbohydrates?
Protection, lubrication, cell-to-cell recognition, adhesion
What are glycocalyx?
Carbohydrates coat on the cell surface
Are the membrane proteins mobile?
Yes
Are microvilli motile?
Not capable of significant mobility
What are specializations of the plasma membrane?
Microvilli, stereocilia
Are stereocilia motile?
Not considered to be motile
What are the membrane transport mechanisms?
Passive transport
Active transport
What happens in passive transport?
Transport depends simply on the difference in the concentration of substances inside and outside the cells
What happens in active transport?
substances move against their concentration gradients and require energy
What are the two processes involved in movement in materials into and out of cells? What is involved?
Endocytosis and exocytosis
involve membrane vesicles and proteins called coat proteins that brace the vesicles
What is endocytosis?
Process by which extracellular materials are captured and enclosed within membrane-ground carriers that invaginate and pinch off into the cytoplasm from the membrane
What is exocytosis?
process where intracellular molecules such as hormones, antibodies, digestive enzymes and matrix-proteins contained within a membrane-bound vesicle discharged outside of a cell by fusion of the vesicle with plasma membrane of a cell
What are the coat proteins involved in endocytosis/exocytosis?
Clathrin, caveolin
What happens in endocytosis?
Membrane invaginates, forming a pocket (containing
macromolecules/other materials from cell exterior)
- mediated by a class of proteins called “coat proteins” that brace the vesicle.
Pocket begins to pinch off, enclosing the extracellular material.
Membrane closes around the invaginated materials, forming vesicle.
Vesicle separates from the plasma membrane, carrying materials from exterior within a membrane derived from the plasma membrane
What are coated pits?
invaginations braced by special membrane based proteins and are used to bring material in to the cell for further processing
What is clathrin?
protein which braces the coated pit membranes.
forms hexagonal lattice structure around the outside of the
vesicle (accounts for the fuzzy coat seen ultrastructurally)
How is the clathrin scaffolding broken down?
special enzymes when the vesicle docks
What are caveoli?
invaginations braced by the protein caveolin
What is potocytosis in caveoli? *
The surface caveoli may carry receptor proteins, which bind to molecules in the extracellular space. They can concentrate substances from the extracellular space and
transport them into the cell
What is transcytosis in caveoli? *
Caveoli used to transport materials from the extracellular space on one side to the extracellular space on the other side
What is signaling in caveoli?
believed to have roles in intracellular signaling.
In many instances special receptor proteins are present in the cell membrane which can bind to specific substances outside the cell and draw them inside in a process termed receptor mediated endocytosis
What is pinocytosis?
nonspecific ingestion of fluid and small protein molecules via small vesicles
What is phagocytosis?
ingestion of large particles such as cell debris, bacteria and other foreign materials
What are the general features of the nucleus? *
one of the most prominent and characteristic features of eukaryotic cells
the site within the eukaryotic cell where chromosomes are localized and replicated and where the DNA they contain is selectively transcribed
What are the major components of the nucleus?
Nuclear envelope, chromatin, nucleolus
What is a eukaryocyte?
Eukaryon means “true nucleus” i.e. cells with membrane-bound nucleus, which compartmentalizes the activities of the cell’s genome - both replication and transcription - from the rest of the cellular metabolism
What are the features of a eurkaryocyte?
Repository for almost all the genetic information
Control center for the expression of the information
Basophilic structure
Number of nuclei vary depending on the cell types
Majority of cells have single nucleus
Multinucleated cells - muscle cells
Shape of the nucleus can vary
What is the nuclear envelope?
nucleus is surrounded by a double membrane, with a space between the two phospholipid bilayers. There are numerous nuclear pore complexes on the nuclear envelope
The nuclear envelope consists of
two unit membranes, the inner and outer nuclear membrane and perinuclear space between them
What is the appearance of the nuclear envelope?
Each membrane is about 7-8 nm thick and has the same trilamellar appearance as most other cell membranes.
What is the appearance of the outer membrane facing the cytoplasm of the nuclear envelope?
closely resembles the membrane of the endoplasmic reticulum, covered with ribosomes and continuous with ER
What is the appearance of the inner membrane facing the cytoplasm of the nuclear envelope? *
supported by a rigid network of protein filaments attached to its inner surface, called nuclear lamina. In addition, the inner nuclear envelope contains specific lamin receptors that bind to chromosomes and secure the attachment of the nuclear lamina
What is the appearance of the perinuclear space facing the cytoplasm of the nuclear envelope? *
filled with fluid and continuous with the ER. The components of the perinuclear space protects and regulates the function of the genome embedded within the nucleoplasm
How is the nuclear pore formed?
by the fusion of the two membranes of the nuclear envelope and is lined with an intricate protein structure called nuclear pore complex (NPC) and provides direct contact between the cytosol and the nucleoplasm
How is the nuclear pore complex visible?
Freeze-fracture microscopy
What does the nuclear pore complex consist of?
100 or more different kinds of peptide subunits
What is the shape/appearance of the nuclear pore complex?
Octoganal arrangement of subunits, protrude into both cytoplasmic and nucleoplasmic sides
Presence of central granule called “the transporter”
Fibers extend from the octagonal ring into the cytosol and nucleoplasm
How does nuclear transport work with the nuclear pore complex?
A “cargo” protein with NLS or NES translocates through nuclear pores bound to its cognate receptor protein.
Importins and exportins are thought to diffuse through the channel, filled with hydrophobic matrix of FG-repeats.
Both transport processes also require participation of Ran, a monomeric G protein that exists in different conformation when bound to GTP or GDP
Analogous to the cytoskeleton of the cytoplasm, there is a structural network of fibers in the nucleus which extends throughout the nucleus
Nuclear lamina
What is the nuclear lamina?
thin electron-dense protein layer which acts as nucleoskeleton analogous cytoskeleton
What does the nuclear lamina contain?
intermediate filament and lamina associated proteins
What does the nuclear lamina do?
lamins disassemble during mitosis and reassemble when mitosis ends.
serves as a scaffolding for chromatin, chromatin-associated proteins, nuclear pore complex and membrane of the nuclear envelope.
lamina is also involved in nuclear organization, cell cycle regulation and cellular differentiation
What is chromatin?
particles or beads called nucleosomes on a double stranded DNA string. Chromatin is in a highly extended state in the interphase nucleus
What does each nucleosome consist of?
histone octamer core and about two turns of DNA wound around the histone core. The histone octamer contains two molecules each of H2A, H2B, H3 and H4 histones
How is chromatin distributed?
Not randomly
Chromatin of each chromosome has its own domain
What is euchromatin?
Dispersed nuclear materials throughout the nucleus and represent active transcription of genetic materials. At the electron microscope level appears less electron dense whitish gray structure
What are the types of heterochromatin?
Constitutive
Facultative
What is constitutive heterochromatin?
Consists of simple sequences of repeated DNA
e.g.centromere and telomere
What is facultative heterochromatin?
Chromosomal regions that are inactivated in a cell.
The amount depends on the activity of the cell
What is the composition of facultative heterochromatin in embryonic and terminally differentiated cells?
Embryonic cells - little facultative Heterochromatin
Terminally differentiated cells- substantial facultative Heterochromatin.
What are nucleoli?
large prominent structures present in every eukaryotic nucleus and also known as the ribosome factory
What are the morphological distinct regions of the nucleolus?
Fibrillar centers
Fibrillar component
Granular component
What are the fibrillar centers of the nucleolus? *
contain DNA loops of five different chromosomes (13, 14, 15, 21 and 22) that contain rRNA genes together with significant amounts of RNA polymerase I and transcription factors
What are the fibrillar components of the nucleolus? *
contains ribosomal genes that are actively undergoing transcription accumulating large amount of ribosomal RNA. The newly synthesized RNA forms an electron dense structure (labeled as fibrils in the adjacent high magnification electron micrograph of the nucleolus)
What are the granular components of the nucleolus? *
represents the site of initial ribosomal assembly and contains densely packed preribosomal particles of 15-20 nm in diameter
What are the types of nuclear proteins?
Histones, non-histone
What are histones?
Small proteins (most are 10-15kD: some up to 28kD)
A single histone complex is actually made of 9 basic histone proteins
What is a nucleosome?
DNA coiled around a histones complex
What are non-histone proteins bound to DNA?
May play many roles in regulation of RNA transcription, and regulation of DNA production, repair, etc.
What is the membrane like in mitochondria?
Double membrane
What is the evolutionary significance of mitochondria? *
evolved from bacteria that were endocytosed into ancestral cells containing eukaryotic nucleus forming endosymbionts
What is the endoplasmic reticulum?
network of membranes throughout the cytoplasmic area of the cell. The ER membranes form complexes to give rise to vesicles and tubules or flattened sacs
What are the types of endoplasmic reticulum?
Rough faced, smooth-surfaced
What is the appearance of rough ER? *
cytoplasm of a chiefly protein synthesizing cell stains intensely with basophilic dyes due to the presence of RNA involved in secretory or membrane bound protein synthesis
What is the appearance of smooth ER?
structurally similar to the rER but lacks the ribosome docking proteins. It tends to be tubular rather than sheet-like, and it may separate from rER or an extension of it. The sER is abundant in cells with lipid metabolism i.e. synthesis of fatty acids and phospholipids
What do the sER in the liver help with? *
detoxifying enzymes modify and detoxify hydrophobic compounds such as pesticides and carcinogens by chemically converting them into water-soluble products that can be eliminated from the body
What is the sacroplasmic reticulum?
In skeletal and cardiac muscle, the sER is also called the sarcoplasmic reticulum which sequesters Ca++, which is essential for the contractile process and is closely apposed to the plasma-membrane invaginations that conduct the contractile impulses to the interior of the cell
What are several ribosomes together form?
short spiral arrays called polyribosomes or polysomes in which many ribosomes are attached to a thread of messenger RNA (mRNA)
What does the golgi complex do? *
glycosylation of the proteins produced in the ER and their packaging.
The site of processing and packaging of ER products, located near nucleus, prominent in secretory cells
What happens with disorders of glycosylation? *
associated with mutations in glycosylation enzymes (found in the Golgi) that cause improper glycosylation of proteins
What are peroxisomes?
small membrane bound, self replicating organelles that contain oxidative enzymes, particularly catalase and other peroxidases. Enzyme content varies with extracellular conditions and tissue types
What do peroxisomes do in the liver?
Involved in the detoxification of ethanol
Where are peroxisomes found?
on all cells and abundantly in the liver and kidney cells. The number of peroxisomes in a cell increases in response to diet, drugs, and hormonal stimulations
What are lysosomes? *
membrane-bound sacs containing hydrolytic enzymes
What do the membranes of lysosomes contain? *
unique proteins (lysosome-associated membrane proteins (lamps), lysosomal membrane glycoproteins, and lysosomal integral membrane protein (limps))
protects the cell from the acid hydrolases contained within. contain proton pumps for the acidification of its contents and number of transport proteins which transport the products of hydrolysis to the cytoplasm
How are lysosomes selectively stained? *
with dyes such as acridine orange or stained cytochemically for acid phosphatase
What are the functions of lysosomes? *
digestion of endogenous or exogenous material
What are the types of lysosomes?
Primary, secondary, residual body
What is a primary lysosome? *
contains one or many enzymes. A virgin lysosome
What is a secondary lysosome?
fusion of one or more primary lysosomes with phagocytized material. Contains “all” the hydrolytic enzymes
What is a residual body?
incomplete digestion by the lysosome. The digested material is excreted via exocytosis, absorbed into the cytoplasm or stored.
(Residual bodies will collect in high numbers in lysosomal storage disorders such as Tay-Sachs disease (a fatal syndrome). These are caused by genetic mutations in the genes responsible for coding lysosomal enzyme)
What is autophagy?
term used to describe the removal or digestion of cell components; e.g., mitochondria and other cell organelles
What are the origin of lysosomes?
Ultimately from the Golgi but receive some of the constituents via endosomes (constitutive pathway)
What is the clinical condition associated with lysosomes?
Lysosomal storage disease results when one or more hydrolytic enzymes is defective, substrate(s) pile up in undigested vesicles
