midterm Flashcards
Histology (def’n):
study of cells, tissue, and organs at the microscopic level
To view things 0.25 μm to 1 mm
use light microscope
to view things .2 nm to 0.25 μm
use electron microscope
transmission and scanning
Steps of Processing Tissue
- fix
- dehydrate
- embed
- section
- stain or immunocytochemical staining or using osmium or gold/metal coating
Process of Fixing is to:
crosslink protein and maintain tissue architecture
Process of Embedding is to:
infiltrate/embed tissue with hard material that can be cut into thin slices
Process of Sectioning is to:
cut thin slices of embedded tissue to let enough light through the sample
Process of Staining is to:
light microscopy - histochemical for reactive chemical groups such as charge (i.e. Hematoxylin and Eosin = H&E)
Process of immunocytochemical staining is to:
i.e. with antibodies for specific antigens
Process of using osmium is for:
transmission E.M. for ultra-thin sections of detailed subcellular structure
Process of gold/metal coating is for:
scanning EM to create a 3D-like image
NOT ACTUALLY 3D
Tissue Definition
a group of similar cells and surrounding extracellular matrix and extracellular fluid (also known as ‘intercellular’)
4 major ‘basic’ tissue types
epithelia, connective tissue, muscle, nervous tissue
Definition of Organs
groups of tissues that act together to carryout specific bodily functions.
-Potency (def’n):
ability of a cell to generate other cell types
definition totipotent
an give rise to all
cell types, example is an ‘embryonic stem cell’
pleuripotent
can give rise to a number of cell types, often in a within a specific developmental lineage/tissue type, example is a ‘mesenchymal’ stem cell that can give rise to all cell types normally generated from mesoderm
Differentiation (def’n)
cell specialization which is determined by differential gene expression.
Stem cell (def’n)
able to self-renew (divide/proliferate) and differentiate.
What determines whether the stem cells will proliferate to make more stem cells or whether they will differentiate?
The microenvironment (eg. the neigbouring cells, the soluble factors present) that stem cells find themselves in (eg. the stem cell niche)
When differentiation occurs..
Potency decreases (during normal development)
induced pluripotent stem cells (iPS)
generated from adult cells by reprogramming them with transcription factors that normally initiate stemness/increase potency (eg. the Oct and Sox transcription factors).
Development (def’n)
Combination of stem cell proliferation and daughter cell differentiation ultimately giving rise to all of the tissues of the embryo.
Fertilization
- occurs in the oviduct
- receptor-mediated process that leads to membrane fusion of sperm and egg
- generates the single cell zygote (totipotent)
Zygote to Morula
- zygote proliferates (mitotic divisions) without differentiating
- generates a solid mass of cells (=morula) that stick together via ‘cell adhesion molecules’
- morula moves down the uterine tube into the uterus proper.
Morula to Blastocyst
- Morula pumps fluid into its center to form a central cavity
- structure now known as the ‘blastocyst’
- significant differentiation
Embryoblast cells
form embryo proper
-during implatation; splits into two layers of cells and differentiate into the epiblast and the hypoblast
Trophoblast cells
help to form the fetal portion of the placenta
the cells are “extraembryonic”
-during implantation; split into two layers which differentiate into the inner, fully cellularized ‘cytotropholasts’ that proliferate, and the outer fused/multinucleate ‘syncytiotrophoblasts’ (= syncytium)
- surrounded the entire blastocyst as it enters the uterine wall
Syncytiotrophblasts
- send finger-like projections deep into the uterine wall that release proteolytic/digestive enzymes that facilitate the implantation of the embryo
- release human chorionic gonadotrophin (hCG) to maintain the corpus luteum in the ovary
Corpus Luteum
- keeps producing estrogen and progesterone
- maintain the uterine wall (ie. prevents menstruation that would lead to the loss of the implanted early embryo)
Trophoblastic Lucanae
- spaces that form within the core of syncytiotrophoblast fingers
- in the placenta;
- > they expand and fuse to form the large ‘intervillous spaces’ that are filled with maternal blood
Epiblast cells
- consists of totipotent embryonic stem cells and amnionic cells
- during the formation of the extraembryonic membrane and placenta;
- > splits and the ‘amniotic cavity’ forms within it
- > the layer of epiblast cells adjacent to the cytotrophoblasts become ‘amnioblasts’
- > gives rise to the extraembryonic mesoderm
Hypoblast cells
- generates the cells of the Heuser’s membrane which delineate the margin of the
yolk sac.
Amnioblast cells
- form the “extraembryonic” amniotic membrane that line the amniotic cavity
Definition Extraembryonic
anything that will not form the embryonic tissues that ultimately give rise fetus proper
Extraembryonic mesoderm
- contributes to all of the major extraembryonic membranes that surround the embryo proper (eg. it contributes to the linings of the chorionic amniotic and yolk sac cavities) and the placenta.
- gives rise the extaembyronic blood vessels that exchange gases, nutrients and waste with the maternal blood in the trophoblastic lacuna.
- migrate out from the epiblast and line the Heuser’s membrane and the cytotrophoblast and surround the embryo proper as well as the yolk sac.
The Placenta consists of:
-Maternal/Uterine component (=’decidua basalis’)
-Embryonic/Fetal component (=’chorionic villi’)
syncytiotrophoblasts, cytoblasts and the cells that line the extraembryonic/fetal blood vessels derived from the extraembryonic membrane
Maternal/Uterine component (=’decidua basalis’) contains:
- mostly maternal arteries and veins which supply and drain the ‘trophoblastic lacuna’
Chorionic Villi
- single chorionic villus is a finger of extraembryonic tissues
- made up of syncytiotrophoblasts and cytotrophoblasts that surround a core of extraembryonic mesoderm to exchange gases, nutrients and waste in the trophoblastic lacuna
The Barrier between the fetal blood and the maternal blood is made up of:
syncytiotrophoblasts, cytoblasts and the cells that line the extraembryonic/fetal blood vessels derived from the extraembryonic membrane
Amnion
- formed from epiblasts which splits and fluid starts to accumulate in the cavity that forms within it. Embryogenesis happens in the amnionic cavity.
- provides a large fluid filled cavity to protect the embryo and fetus; helps eliminate waste.
Yolk Sac
- formed from hypoblasts that migrate along Heuser’s membrane which first delineates the fluid-filled yolk sac.
- part breaks off later on and is reabsorbed.
- a transient structure that produces blood cells until the liver forms; germ cells form in the yolk sac and migrate to the gonads
Chorionic Cavity
- forms between (What layer??) two layers of mesoderm as fluid fills in gaps.
- it becomes a potential space, and amnion replaces this space as it expands through the embryonic period and, especially the later fetal period.
How amniocentecis works
A small number of cells sluff off into the amniotic fluid
- the cells can be removed from the fluid and their chromosomes/genes can be analyzed
Definition Gastration
Gastrulation is the conversion of the BILAMINAR (epiblast and hypoblast)
embryo into three germ layers (ectoderm, mesoderm, endoderm).
Primitive Streak
- Formed from the epiblast
- starts at the caudal/tail end of the embryo
- as the epibalst cells divide/proliferate the lateral edges of the streak they start to push upward and the cells at the centre ingress
- sets the axes of the embryo
- the anterior portion is called the primitive node which moves toward what will form the cranial/head end of the embryo
The beginning of Gastrulation is marked by:
- the formation of the primitive streak, the primitive node
epithelial to mesenchymal transition (EMT)
- the cells that ingress into the primitive streak start to express novel sets of genes (differentiate).
- causes the cells to cease being ‘epithelial’ and become single and migratory = ‘mesenchyme’
Formation of Endoderm
- first wave of mesenchymal cells push down into, and replace, almost all the cells of the hypoblast and become the endoderm
Endoderm gives rise to:
-many of the innermost lining tissues of the body (ie. much of the GI, Respiratory and Urinary tracts).
Formation of Mesoderm
- 2nd wave of mesenchymal cells push between the epiblast and hypoblast to form the mesoderm
- early on different regions regulate the induction of tissues and structures
Mesoderm gives rise to:
-most of the ‘packing’ tissues of the body (ie. muscle, bone, cartilage, connective tissue, blood, fetal/post-natal blood vessels)
Formation of spinal cord and brain
- the notochord formed from the mesoderm induces the formation of the neural tube
- the precordal mesoderm moves cranially and induces the formation of the portion of the neural tube that forms the brain
- Formation of the brain requires the expression of the transcription factor Lim1, with out Lim1 there is no head and brain formation
Paraxial mesoderm forms:
- segmented “somites” that generate skeletal muscle, cartilage, and tendons as well as much of the dermis
Formation of Ectoderm
- cells that don’t enter the primitive streak and remain in the top layer of the developing embryo form the ‘ectoderm’ which gives rise to most of the outer covering tissue of the body (ie. skin) and the nervous tissue (by ‘induction’; see below).
Ectoderm gives rise to:
- most of the outer covering tissue of the body (ie. skin) and the nervous tissue (by ‘induction’)
Formation of the Notocord
- the last mesenchymal cells to move through the primitive streak aggregate to form a solid cord of mesodermal cells in the midline = ‘notocord’
- located just below the ectoderm that will form the spinal cord.
Definition Neurulation
Formation of the neural tube, done by the process of “induction”
Definition Induction
- process whereby one cell or group of cells influences the developmental fate of another.
e. g. the Notochord and prechordal mesoderm induce the overlying ectoderm to form nervous tissue.
Formation of the Neural Plate
- notochord releases short range-acting molecules that induce formation of the central nervous system (CNS) by signaling the ectoderm directly above it to thicken
Neural Groove
- neuroectodermal cells of the neural plate change shape such that it folds in upon itself to form the ‘neural groove’.
- These cells change their cell-cell adhesion molecules to a neural form (eg N-cadherin) which is different from those expressed in remainder of the ectoderm (eg. E-Cadherin).
- allows the tissues to separate from each other.
Neural Crest
- A small number of the N-cadherin producing neuroectodermal cells pinch off from the developing neural tube and move laterally to form the neural crest
Formation of the 3 mesodermal regions
- the neural plate buckles inward, the mesoderm alongside the spinal cord portion of the neural tube differentiates to form the three mesodermal regions:
1) paraxial (somites, forming in a cranial to caudal fashion)
2) intermediate
3) lateral plate
Somites
- segmented blocks of mesoderm on each side of the midline give rise to most of the skeleton and all the voluntary musculature.
Specialized Patterning of the Neural Tube
- neural tube forms specialized regions to produce different cell types ex dorsal plate for sensory information and ventral plate for motor information
- the developing spinal cord portion of the tube ‘positional cues’ come initially from two regions:
1) Notochord
2) Dorsal Ectoderm Cells - the combination of chemical gradients provide ventral/dorsal spatial information across the neural tube which induces different combinations of transcription factors to be expressed in different spatial domains that cause differentiation in those domains
During specialization of the Neural Tube the Notochord releases
- Sonichedgehog (Shh) which diffuses and decreases in concentration as it moves dorsally
Definition Transcription Factor
binds DNA at specific sequences to turn on/off specific genes in clusters of cells that act as ‘progenitors’ which have stem like characteristics.
During specialization of the Neural Tube the Dorsal Ectoderm Cells release
- Bone Morphogenetic Protein (BMP) which diffuses and decreases in concentration as it moves ventrally
Trilaminar nature of the Cell Membrane
- outer polar heads - dark on TEM
- middle hydrophobic tails - light on TEM
- inner polar heads - dark on TEM
4 ways proteins associate/interact with membranes
- Integral membrane proteins spans the phospholipid bilayer
- Covalently-linked to a fatty acid tail (e.g. palmitoylation) that inserts in membrane
- Covalently-linked to a specialized phospholipid (eg. glycophosphatidylinositol-GPI) that
inserts in the membrane - Peripheral proteins that associate with an integral membrane protein (i.e. in close proximity
to, but not inserted into the phospholipid bilayer)
The GPCR-cAMP receptor
GPCR
The GPCR-cAMP transducer
G-protein
The GPCR-cAMP amplifier
Adenlyate cyclase
The GPCR-cAMp messenger
cAMP
The GPCR-InsP3 receptor
GPCR
The GPCR-InsP3 transducer
G-Protein
The GPCR-InsP3 amplifier
PLC which turns PtdIns4,5P2 into the messenger
The GPCR-InsP3
InsP3 and diacylglycerol
Endoplasmic reticulum
- site of biogenesis of other organelles
- RER Functions in protein synthesis and post-translational modification (glycosylation, adding
disulfide bonds and protein folding) and SER is important in lipid synthesis and intracellular calcium
storage. - Also site of many detoxification enzymes, particularly in liver hepatocytes.
Protein Synthesis in the RER
signal sequence’ = N-terminal portion of growing peptide, which binds to the….
• ‘signal recognition particle’ in the cytoplasm, which binds to the…
• ‘signal recognition particle (SRP) receptor’ on the ER membrane which contributes to the formation
of a channel that threads growing peptide into the ER lumen where the….
• ‘signal peptidase’ removes the signal sequence from the polypeptide in the ER lumen and…
• ‘post-translational modifications’ occur including protein folding, disulphide bond formation between
strands of the protein, and glycosylation of the protein. These steps occur in the ER lumen to generate a ‘completed protein’ for packaging and eventual export from the ER to other cellular sites or secretion from the cell
Unfolded protein response’ = UPR, which is manifested in three ways:
i) ER-associated degradation (ERAD) of the misfolded protein; specifically, it is removed from the ER and destroyed in lysosomes or the proteosome (see 05Lect Cell II)
ii) An upregulation of the ER machinery that facilitates protein folding (induces the production of protein chaperones and lipid synthesis that associate with and help proteins fold)
iii) ‘Apoptosis’; this is the last resort whereby, if large amounts of misfolded protein cannot be destroyed or re-folded, the cell initiates a program that ultimately leads to cell death (also known as ‘programmed cell death’)
Cystic Fibrosis - An example of UPR causing a clinically relevant problem
•mutations in the Cystic Fibrosis Transmembrane Regulator gene, which codes for an integral transmembrane protein that is a chloride channel, prevent proper protein folding and initiate a UPR which reduces transport of the CFTR channel from ER to the plasma membrane and increases its destruction by ERAD.
- Loss of CFTR causes Cystic Fibrosis*
- treatment:
- > gene therapy (delivering the wild-type gene via viruses)
- > pharmacologically suppress the UPR to increase delivery of the protein b/c some are still functional even with the mutations
Golgi
- ‘cis’ face that receives vesicles from the ER and a ‘trans’ face that releases mature vesicles to go to various locations in the cell
Vesicular transport model
- cargo moves through the Golgi via small, spherical vesicles that bud off an individual cisterna and move to the next cisterna in the stack. - In this model the Golgi itself would be static while the cargo is dynamically moved through it in vesicles.
Cisternal maturation model
- the cisternae themselves move through the stack carrying cargo along with them
- in this model the small vesicles bud off and travel back down the stack (i.e. back towards the cis face) to recycle the Golgi enzymes so that they can work on the next wave of cargo that arrives from the ER.
types of endocytosis
- Phagocytosis, Pinocytosis, Receptor-mediated
- counter act exocytosis
Trafficking and Sorting in the Golgi
ex lysomomal pathway
- Lysosomal enzyme (= cargo molecule), which needs to be trafficked/sorted to the
lysosome, is phosphorylated on mannose to generate ‘mannose-6-phosphate’ (M6P) in
the cis-Golgi. - M6P binds to the ‘M6P Receptor in the trans-Golgi which recruits the coat protein
clathrin. - A vesicle containing the enzyme-receptor complex buds from the trans-Golgi.
- The vesicle uncoats and is then targetted to and fuses with the late endosome.
5/6. The acidic pH of the late endosome releases of the lysosomal enzyme from the M6P
receptor.
7/8. The free M6P receptor is then recycled back to trans-Golgi, while the lysosomal enzyme
is dephosphorylated and traffics to the lysosome where it is now active (i.e. due to the low pH and dephosphorylation).
receptor-mediated endocytosis pathway
1.The secreted lysosomal enzyme binds M6P receptor on the plasma membrane.
2.The enzyme-receptor complex recruits clathrin and an endocytic vesicle forms that buds
into the cytoplasm
3/4. The vesicle uncoats and fuses with an early endosome, which then fuses with a late
endosome.
5. The acidic pH of the late endosome releases the lysosomal enzyme from the M6P
receptor and dephosphorylates the enzyme itself.
6/7/8. The M6P receptor then recycles back to either trans-Golgi or the plasma membrane;
the dephosporylated, active lysosomal enzyme is trafficked to the lysosome.
Tay-Sachs disease
Doesn’t have the Hexosaminidase A (Hex-A) enzyme, which acts to break down specific types of lipid (eg. ganglioside GM2). Therefore, these lipids accumulate abnormally in lysosomes to toxic levels, especially in nerve cells in the brain.
Xanthomas
- Deposits of cholesterol in skin.
- Occurs in patients with a deficiency in Low Density Lipoprotein receptors (LDL normally
carries the lipid cholesterol in blood). This most often occurs because the LDL receptor is
not trafficked to/recycled from the plasmamembrane correctly. - Because of this deficiency, cells can’t take in LDL and can’t catabolize cholesterol, resulting in high levels of circulating cholesterol in the blood (hypercholesterolemia) and lipid/cholesterol deposits/swellings in the skin = xanthomas.
Lipid Traffic
-Lipids can be trafficked either:
1) In vesicles (ie. within the membranes of vesicles that directly fuse, as described above, with
other vesicles, with other membrane-bound organelles, or with the plasma membrane).
2) In a non-vesicular manner hidden within lipid transfer proteins (LTP’s) that are soluble in
aqueous solution, most often across short cytoplasmic gaps between membranes that are in close contact with each other.
Classes of lipids
- Glycerolipids: has a glycerol backbone, and a hydrophilic choline as well as two hydrophilic fatty acids
- Sphingolipids: has a sphingosine instead of a glycerol backbone; some still have a choline attached like glycerolipids, while others have a sugar attached; as well as one fatty acid
- Sterols: e.g. cholesterol
Cholesterol Transport via LTP
- LTP binds to receptors on donor membrane and loads cholesterol into the barrel of the LTP.
- LTP dissociates from the donor membrane and diffuses to the acceptor membrane.
- LTP binds the acceptor membrane via receptor, into and unloads the cholesterol into the
acceptor membrane. - LTP dissociates from the acceptor membrane so that it can used to transport another lipid
non-vesicular lipid traffic
- lipids are transported from one membrane to another across a cytoplasmic gap at defined sites where the membranes are very close together (10-50nm), the gaps are know as ER Junctions (ERJ)
- transported inside the aqueous soluble LTP’s which have hydrophobic pocket/barrel to hide the lipid from the cytoplasm
- there are many families of LTP’s which carry specific lipids to and from various structures in the cell, either for transport or for lipid synthesis/modification
ER Junctions
- for non-vesicular lipid traffic
- formed by ‘bridging complex’ proteins that bind the membranes of both structures. The membranes come so close together in ER junctions that the LTP’s are able to bind to both the donor and acceptor membranes simultaneously for highly efficient transfer between the two membranes
- grab and release lipids as they use their flexible ‘hinge to swing between the donor and acceptor membranes
Mitochondria
- The mitochondrial circular DNA (cDNA) contains only 37 genes, but the mitochondrion has
~1000 proteins; Therefore, most mitochondrial proteins are coded by nuclear genes and are
imported into the mitochondria.
-Mitochondrial import occurs at outer/inner membrane contact sites (eg. the components
involved are brought in close contact such that the transfer of the protein occurs over a short distance without membrane fusion). - impaired mitochondrial structure, which leads to impaired energy production, causes mitochondrial myopathy whose clinical signs (poor muscle control/posture and cognitive impairment) are mostly the result of globally inefficient ATP production in cells with high metabolic rates (eg muscle cells and neurons).
Stages of Mitochondria Import
ex ATP synthase
I) Chaperones bind the polypeptide to be imported in the cytoplasm and transports it to the
mitochondrion.
II)A mitochondrial targetting sequence in the polypeptide directs it to the Translocon of the
Outer Membrane (=TOM) complex which is a large transmembrane channel that drives the polypeptide through the outer membrane into the intermembrane space of the mitochondrion.
III/IV) The small Translocon of the Inner Membrane (=TIM) complex, which is a peripheral membrane, binds the polypeptide delivers to the large TIM complex, which is a large transmembrane protein complex that inserts the polypeptide into the inner mitochondrial membrane membrane.
V) Polypeptide is released into the membrane and it folds into its native structure.
Peroxisomes
- Small, membrane bound organelles; self-replicating; important in regulating metabolism in all cells (eg. ß-oxidation-mediated breakdown of fatty acids which generates Acetyl-CoA that is utilized in the citric acid cycle in the matrix of the mitochondria).
• All proteins are trafficked/transported to peroxisomes as they have no genetic material via: 1) A peroxisomal targeting signal sequence in the protein that targets cytosolic proteins to the peroxisomal membrane in a manner similar to protein targetting to the mitochondria;
2) From the ER by fusion with ER-derived vesicles.
• Peroxisomal defects affect many tissues, often severely (eg. Zellweger’s Syndrome is caused by an impairment of peroxisomal protein import that causes death shortly after birth).
Cytoskeleton
Form a 3D network of protein filaments that act to maintain cell morphology, move intracellular components (e.g. vesicles and organelles), and facilitate the movement/ migration of entire cells
- 3 main classes
Actin
~6nm diameter
• ‘Thinnest’ cytoskeletal element; formed of filaments called F (fibrous)- actin which are
comprised of two chains of globular actin monomers (= G actin).
- filaments are dynamic with plus (faster growing) ends and minus (slower
growing) ends.
- Capping proteins regulate the length and speed of growth.
- Filaments can be bundled together for different functions, and bundles are maintained by
different actin filament binding/stabilizing/cross-linking proteins (eg. α-actinin)
- Myosin motors can attach to F-actin to either initiate contraction (both in muscle and non-
muscle cells) or move cargo (vesicles).
- Focal adhesions are regions where the actin cytoskeleton binds/adheres to the extracellular
matrix (ECM) across the plasmamembrane (Fig 2.32).
• F-actin in ‘stress fiber’ bundle of f-actin are linked to the cytoplasmic domains of
transmembrane integrins by a scaffold of stabilizing proteins (eg. vinculin and talin).
- The extracellular domains of the integrins then bind extracellular matrix molecules (eg fibronectin, laminin or collagen).
- These attachments are critical for cell adhesion and migration (ie. during embryogenesis, tissue formation, wound-healing and many organ system functions).
- can also be linked together into gel-like webs and networks by ‘scaffolding’ proteins such as cortactin and filamen in the outer portion/cortex of the cell. These actin networks are critical for cell shape and, therefore, are very important for the form and function of tissues.
