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.
Intermediate filaments
(8-10nm in diameter):
• Filaments are ‘intermediate’ in diameter and formed by overlapping protein rods.

• Form structural scaffolds within the cytoplasm (eg. cytokeratins) and the nucleus (eg. lamins)
Microtubules
(25 nm in diameter):
• Hollow tubes comprised of 13 protofilaments fromed from globular α- and β- tubulin
heterodimers that have a ‘+’ (plus) ‘growing’ end where dimers can be added to increase the
length of the tubule
• Originate at the centrosome from γ-tubulin ring complexes = the ‘-‘ (minus)
o Centrioles are specialized microtubular structures within centrosomes, nucleate microtubules and generate the long spindles that attach to chomosomes during mitosis
• Microtubules also critical for movement of organelles in the cell, and for the movement of specialized cellular projections (eg. cilia and flagerlla) as well as chromosomal segregation
• ‘Kinesin’ motor proteins bind cargo and move along microtubules towards the plus end, whereas ‘dynein’ motor proteins move toward the minus end of the microtubules.
Nucleus
- nuclear structure: euchromatin (light patches in figure, unwound DNA that can be effiently transcribed); heterochromatin (densely packed, not being transcribed); nuclear envelope; and nuclear pores, which are large protein assemblies embedded in the nuclear envelope;
• Nuclear pores have a size of 9-11nm and allow small molecules to pass freely in and out of the nucleus. Larger molecules must be tranported in (import) and out out (export) of the nucleus. Thus, the number of nuclear pores correlates with the metabolic activity of the cell
process of nuclear import and export
- The major concept here is that the RanGTPase, is maintained in two different states depending on location: in the cytoplasm it is GDP-bound (‘off’); in the nucleus it is GTP- bound (‘on’)
- In the cytoplasm cargo proteins with a ‘Nuclear Localization Signal’ (NLS) interact with the importins which leads to their movement through the nuclear pore as a complex into the nucleus
- Once in the nucleus RanGTP binds the cargo/importin complex (via importins’ ‘Nuclear Export Sequence [NES] and the cargo is released; RanGTP/Importin are then exported out of the nucleus
- In the cytoplasm the ‘GTPase activating proteins (‘RanGAPs’) convert RanGTP to RanGDP. This turns the Ran ‘off’ such that it releases the importin proteins so that they can begin the cycle again with new cargo proteins
- RanGDP, which is small in size diffuses back through the nuclear pore. In the nucleus, Ran Guanine Exchange Factors (RanGEFs) convert RanGDP to RanGTP so that it is once more ‘on’ and ready to export importins (or any other proteins in the nucleus with an NES sequence) once again.
DNA Damage Checkpoint
DNA damage (ie. genetic mutation) activates the portin ‘p53’ which then induces production of the p21 protein that then inhibits the CyclinE/CDK2 complex such that the cell does not move from G1 to S. This gives the cell a chance to repair the DNA damage/mutation. If it cannot, p53 can then direct the cell to undergo apoptosis via the ‘intrinsic’ pathway.
Apoptosis
• Active ‘programmed cell death’
• Reduces inflammation due to debris release that occurs after unprogrammed, passive death
by ‘necrosis’
• Important in controlling cell numbers in development (e.g. cell removal during toe and
finger formation in the hands and feet) and in responding to cellular damage or pathological
processes by clearing damaged cells (eg. tissue damage).
• Two major types of Apoptosis
Extrinsic Pathway of Apoptosis
- initiated by extracellular death ligands (ie. come from outside the cell and therefore are ‘extrinsic’) that bind to death receptors and initiate signaling pathways that activate the executioner caspases that cause a breakdown of the cytoskeleton, membrane blebbing and chromosomal digestion cleavage and cell death.
Intrinsic pathway of Apoptosis
- initiated by intracellular stresses such as DNA damage (and therefore occur inside the cell = ‘intrinsic’) that initiate the release of cytochrome c from the mitochondria that activates the ‘apoptosome’ that ultimately activates the executioner caspases cytoskeletal breakdown, membrane blebbing, chromosome digestion and cell death.
- There is some cross talk between the two pathways such that the activation of one pathway can ultimately activate the other pathway.
cell junctions
- Cell junctions facilitate the formation of tissues by aggregating individual cells with each other and their associated extracellular matrix (ECM)
• Cell junctions are an especially prominent feature of epithelial cells. All of the examples in this lecture are found in epithelia - Cell junctions occur at points of cell-cell or cell-ECM contact
hree ‘functional’ types of cell junctions
• Anchoring - mechanically attach cells to other cells or the ECM
• Occluding - seal the contacts between neighboring cells
• Channel-forming/communicating - form channels between cells for communication/allow
chemical and electrical signals to pass from cell to cell
All junctions are multi-protein complexes containing 3 main types of proteins:
- transmemebrane adhesion protein
- adapter protein
- cytoskeletal linkers
Transmembrane adhesion protein
These are integral membrane receptors that span the cell membrane to connect the inside and outside environment of the cell. The outer extracellular region attaches to other adhesion proteins on neighbouring cells (cell-cell adhesion) or extracellular matrix molecules (cell-ECM adhesion). Their inner intracellular regions attach to cytoplasmic ‘adapter’ proteins and connect to other proteins like cytoskeletal linkers and cytoskeletal proteins themselves (eg. actin). Transmembrane proteins are classified by how many times they cross the membrane: single pass, two pass, three pass transmembrane protein, etc.
Adapter proteins:
They bind to the adhesion complex at the membrane, recruit additional components to the adhesion complex and regulate the adhesion complex.
Cytoskeletal linkers
physically link between the adhesion complex proteins to the cytoskeleton
Three common features of cell junctions that increase their stability and strength
- Made up of multi-protein complexes
- ‘Clustering’ of the transmembrane adhesion proteins in the plasmamembrane - the
combined strength of multiple bond interactions increases overall strength of the junction
that binds to either other cells or the extracellular matrix - The clustered adhesion proteins link to the cytoskeletal network inside the cell which
produces a huge tension-bearing protein interaction network running through the tissue.
Definition Cell Polarity
Positional asymmetry (eg. regional differences) within the cell
Epithelial Cells have 3 regions:
- apical
- lateral
- basal
Apical region of Epithelial cells
this is the free, unattached plasma membrane region that faces an open space (eg. ‘outward’) that may be air-filled (eg. parts of the respiratory tract) or fluid- filled (eg. blood).
Lateral Region of Epithelial Cells
this is the membrane region that is in close contact with neighbouring cells within the epithelium
Basal Region of Epithelial Cells
his is the domain that is attached to extracellular matrix that often faces underlying connective tissues (eg. ‘inward’)
‘Freeze fracture’ electron microscopy
carbon/platimum ‘casts’ are made of frozen membrane surfaces, make it possible to view adhesion protein clusters embedded in the membrane as well as their associated adaptor proteins and linker proteins. For examination purposes in this course you will not have to distinguish between ‘E’ and ‘P’ faces on a freeze fracture electron micrograph.
4 Specific Types Cell-Cell Junctions:
- Tight Junctions/Zonula Occludens
- Adherens Junctions/ Zonula Adherens
- Desmosomes/Macula Adherens
- Gap Junctions/Communicating Junctions
Tight Junctions/Zonula Occludens
- belt that goes all the way around the cell.
• Tight junctions are located very near the apical domain of polarized epithelial cells and they join
neighbouring cells very closely together.
• Formed by strands of interacting transmembrane proteins observable by freeze-fracture electron
microscopy. Two core tight junction proteins are Claudin and Occludin. Specifically, tight junctions are formed by the transmembrane adhesion proteins Claudin (for tight junction formation) and ‘Occludin’ (for barrier function, but not needed for maintaining the overall tight junction structure) binding to the same type of molecule on neighbouring cells (i.e. Claudin- Claudin or Occludin-Occludin interactions).
• Barrier permeability can be tested by using a dye/tracer that is added to either the apical or basal side of the epithelium and observing whether the dye/tracer diffuses over the junction
• Tight junctions control diffusion of material between cells (ie. along the lateral membranes). This is very important physiologically as it sets up epithelia as ‘barriers’ where transport of molecules (eg. glucose for example, across the intestinal epithelium) can be highly regulated through the cells based on polarized insertion of specific plasma membrane transporters in the apical and basal domains
Clinical Relevance of Tight Junctions
Loss of the tight junction adhesion protein Claudin-16 (due to mutation of one of many claudin genes) disrupts the kidney’s ability to efficiently transport electrolytes out of the urinary filtrate. As a result calcified deposits form within the kidneys (= white dots in the CT scans shown in the lecture powerpoint) which can eventually lead to blockage and kidney failure.
Adherens Junctions/ Zonula Adherens
Form strong continuous adhesion belts around the cell (covering the entire circumference) on the lateral domain just basal to tight junctions
• Important for the formation of 2-dimensional sheets of epithelial cells than line our body compartments.
• They are composed of single-pass transmembrane adhesion proteins called ‘classical cadherins’.
Clustering of Cadherin
- At low calcium levels two cadherin molecules interact with each other on the surface of the same cell to form ‘cis-homodimers’
- Calcium-binding to the extracellular domains of the cis-homodimers causes them to undergo a conformational change and ‘straighten’
- Cadherin straightening promotes “trans” homodimerization, which means that a cadherin cis-homodimer on one cell then binds another cadherin cis-homodimer located on the surface of another cell (ie. this initiates adhesion).
- This trans-bound complexes cluster together in the plane of the membrane (ie. this starts to stabilize adhesion).
• Cadherin clusters then link to the actin cytoskeleton via intracellular anchoring complex containing adapter and linker proteins called “catenins” (these strengthen the adhesion between cells).
Clinical Relevance of Adheren Junctions
A loss of cadherins can contribute to the formation of, and the metastatic progression of, cancers that arise in epithelium (= ‘carcinomas’). A hallmark of a carcinoma is the loss of epithelial structure which is often associated with the loss of cadherin (by mutation and/or transcriptional repression). This facilitates a loss of cell cycle control that causes the inital ‘primary’ tumor to form. A loss of cadherins can also facilitate the dissociation of tumor cells from each other which helps initiate ‘tumor invasion’ which is the first step in metastasis that ultimately leads to the progressive formation of tumors at seconday sites that is a major cause tumor mortality. The process of carcinoma-associated cell dissociation and invasion has many things in common with the epithelial-to-mesenchymal transiition [EMT] that occurs when mesenchymal cells to break away from the epiblast and invade the space between the developing ectoderm and endoderm during gastrulation).
Desmosomes/Macula Adherens
- Macula = spot; these are small spotlike-junctions that form along the entire lateral domain of cell types in tissues that are exposed to tensile forces/mechanical stress like skin
• Desmosomes are junctions that are based on adhesions between cadherin-like receptors called Desmocollin (Dsc) and Desmoglein (Dsg), which bind to cytoskeletal linkers to connect with the keratin family of intermediate filaments. Keratin intermediate filaments are stronger than the actin filaments found in adherens junctions.
Clinical Relevance of Desmosomes
Pemphigus vulgaris - A loss of dsg causes a loss of adhesion between layers of the skin, resulting in skin blistering.
Gap Junctions/Communicating Junctions
Major function is to allow communication and sharing of small molecules (eg. ions, signal transduction membranes and some nutrients) between neighboring cells
• Connexin are the gap junction adhesion proteins. Six connexins cluster together as a hexamer called a connexon in the lateral membrane domain of one cell membrane. A connexon will then bind a similar hexamer/connexon in the lateral membrane domain of a neighbouring cell to form a functional channel between the cells that can be regulated (ie. can be opened or closed) and can pass small molecules between linked cells in the tissue (=communication).
Clinical Relevance of Gap Junctions
Vohwinkel Syndrome (keratodema) - Loss of connexin-26 (= one of many isoforms of connexin) causes a disruption of communication between epidermal cells in the skin. This causes the skin to overproduce keratin and thus it becomes hard and thick (= keratodema).
Extracellular Matrix (ECM)
secreted by cells into their outside environment. It is critical for cells to organize into tissues and it contains significant amounts of proteins and complex polysaccharides, and has multiple functions.
Connective Tissue ECM consists of:
- Ground substance
- Fibers
Ground Substance is made of:
- Glycosaminoglycans (GAGs)
- Proteoglycans (PGs)
- Glycoproteins (GPs)
Glycosaminoglycans (GAGs)
- These are chains of carbohydrates (=glycan) that are negatively charged. Their negative
charge attracts cations, particularly sodium, which in turn attracts large amounts of fluid that gives the ground substance its gel-like quality that is resistant to compression.
Heparin
a GAG that is an anticoagulant
Hyaluronic acid (HA)
- a huge and abundant GAG, and it is the only GAG that does not bind proteins directly; it is lubricating and thus it is found in joints; it is also
important in wound repair, cell migration, and inflammation
Clinical Relevance of GAGs
Mucopolysaccharidoses (MPS) diseases; Pleiotrophic meaning they can
can have many effects including dwarfism, mental retardation, cardiomyopathy; Are caused by a failure to break down/remodell GAGs which occurs constantly in the connective tissues normally.
Proteoglycans (PGs)
- These are proteins with many long, unbranched GAG chains attached to them
- The attached GAG chains make up most of the molecule’s mass (up to 95% carbohydrate by
weight) - Aggrecan is a proteoglycan that is a major component of cartilage that serves to aggregate
GAGs, other proteoglycans, glycoproteins, and fibers (eg. collagen) to form a deformable molecular meshwork in the specialized ECM of cartilage
Glycoproteins (GPs)
- proteins with numerous short, branched oligosaccharide chains added to them. They are 1 - 60% carbohydrate by weight. Therefore, most of their mass is usually the protein itself (ie.
there is less sugar attached to GPs than PGs) - Fibronectin is a glycoprotein found in many ECMs, and is a key molecule in blood clotting
and tissue repair - Laminin is a glycoprotein that is prominent in Basement Membrane ECM’s that associate with epithelia specifically
Fibers of Connective Tissue
- long polymers of proteins, are embedded within the ground substance; they can be seen running through the ground substance of the ECM in electron micrographs or with special staining in light micrographs. They are mainly composed of two proteins: collagen and elastin in connective tissue ECM. Fibers function to provide structural support.
Collagen
It’s the main ECM protein; makes up about 25% of proteins present in the body
• It’s flexible, but strong and resistant to stretching
• A collagen polymer is composed of repeating units of tropocollagen, which is a helix of
three α-chain subunits.
- There are 29 different collagen genes in humans
-Inelastic/don’t stretch; critical for tensile strength of connective tissues
Elastin
stretchy/distensible and flexible, and is the main component of structures known as elastic fibers. In ECM’s where elastin fibers are prominent this gives the tissue the ability to ability to change form under tensional loads and then return to the original form when that load is removed
Connective Tissue
- made up of an extensive mesh-like network composed of interlinked GAGs, PGs, GPs and fibers.
- contains fibroblasts, which synthesizes and secrete the precursors of all components of ECM and collagen fibres, and adipocytes, chrondroblasts, osteoblasts, tendon cells, etc.
Basement Membrane ECM
• Specialized ECM that forms a carpet-like structure located at the interface between epithelia and the underlying connective tissues
• It is a mechanical barrier between epithelial and connective tissues, an anchoring point (firm and flexible support) for the epithelial cells that attach to it via ‘hemidesmosomes’ (see below), and a molecular filter for the epithelium.
• It consists of two parts:
1) Basal Lamina
2) Lamina Reticularis
Basal Lamina
- produced by the epithelial cells; it consists of two layers:
1) Lamina Lucida
2) Lamina Densa - Laminin binds collagen and integrins and acts as a bridge between the two layers
Lamina Lucida
Contains mainly the GPs laminin (ECM glycoprotein that binds to both integrins and fibers of the lamina densa) and integrins (ECM-binding adhesion receptors, see below). This is the ‘clear” portion of the basement membrane on transmission electron micrographs
Lamina Densa
A mixture of fibers (mostly type IV collagen), the PG ‘perlecan’ and the GAG ‘heparin sulfate’ that form a network that gives the basement membrane carpet its tensile strength. This is the ‘dark’ (eg. electron dense) portion of the basement
membrane on transmission electron micrographs.
Lamina Reticularis
• Produced by connective tissue cells
• Contains collagen I and III, the GP ‘fibrillin’ and anchoring fibrils (type VII collagen fibers).
These anchoring fibrils reach up into the basal lamina above and down into the connective
tissue ECM below to hold things together
• Can be thought of as the spiky/sticky/tack-like bits of the basement membrane that holds
the carpet onto the underlying connective tissue ECM
• Clinical relevance: Alport syndrome - caused by mutations in collagen IV and therefore
affects basement membrane structure; becomes progressively worse with age leading to kidney failure, hearing loss, and cataracts.
Cell-ECM Adhesions = Cell/Integrin Adhesion Complex
• These structures link/attach cells to the ECM
• Their are overall molecular architecture is similar to cell-cell junctions (see Lecture 7) in
that they consist of:
• Cell adhesion molecules
• Linker/adapter molecules that bind both the adhesion molecules and the cytoskeleton
• Cytoskeletal molecules
• The difference, however, is that the cell adhesion molecules (the main one being integrins) bind to ECM glycoproteins and fibers rather than cell adhesion molecules on other cells.
Integrins
• Transmembrane/integral membrane protein ‘heterodimers’ that are made up of two different subunits designated ‘alpha’ (18 in humans) and ‘beta’. (8 in humans)
• There are many different combinations of alpha/beta dimers which gives individual integrins a specificity for binding to many different ECM ‘ligands’ that include fibers (eg. collagens) and glycoproteins (eg. laminin, fibronectin) and, in the case of activated platelets, ‘fibrin’ in clotted blood
• Integrins exist in two states: low affinity and /high affinity states with respect to ECM binding.
• Integrins are activated in two ways: a) “outside-in”: by the binding of ECM ligands just outside the cell which pulls on the integrin and opens it up to the active state, or b) “inside- out”: by integrins binding proteins inside the cell that pull on them and move them into a high affinity state
- these shifts between inactive and inactive states are ‘tuneable’ between the two states
• After ligand binding integrins can also cluster just like other cell adhesion molecules do -
see slide titled ‘The integrin adhesion complex’. This increases the strength and stability of
the cell-ECM adhesion.
• Functions: cell migration; cytoskeletal organization, signal transduction modification;
attachment of tendons to muscles; ligaments to bone; attachment of epidermis to dermis in the skin
Focal Contacts = Focal Adhesions
- Link to actin
• These adhesions are found in many cell types
• Made up of a variety of integrin heterodimers
• They are often very dynamic and they can be used to generate traction forces on the ECM
(ie. can ‘pull’ on it due to the actions of motors such as myosins on bundles of actin filaments called ‘stress-fibers that are attached to these adhesions). Therefore, focal adhesions are critical for cell movement/migration.
• Stable forms of these adhesions are important for muscle attachment to tendons (which are made up mostly of collagen fibers; see Lectures 12/13). This relationship has been examined experimentally in ‘fruit flies’ (Drosophila), a model organism where the individual molecular components of the muscle/tendon attachment can be easily manipulated genetically
Hemidesmosomes

- Link to intermediate filaments
- These specialized adhesions are found in epithelial cells exclusively.
- Make up of α6β4 integrin heterodimer
- They bind the basal surface of polarized epithelial cells to laminin in the underlying lamina lucida of the basement membrane ECM. Thus, they are very stable, anchoring junctions. The stability and anchoring function of these junctions is facilitated by the interaction with strong and stable intermediate filaments. The stability of these junction is also facilitated by the extremely long intracellular/cytoplasmic tails of alpha6/beta4 integrins.
- On a transmission electron micrograph hemidesmosomes look like ‘half a desmosome’, which is how they originally got their name.
Clinical Relevance of Hemidesmosomes
‘Junctional epidermolysis bullosa’ is an extreme skin blistering disease, that is caused by the loss of β4 integrin. Thus, epithelial cells (ie. skin keratinocytes) cannot anchor properly to the underlying laminin in the basement membrane. As a result the epithelium of the skin lifts and blisters when any tension or extreme movement is applied to it.
Epithelium Characteristics
a. Continuous layer(s) of cells stuck together with no extracellular matrix between them. Connection are
mediated through cell-cell junctions such as desmosomes, tight junctions and gap junctions
b. Attached to the basement membrane through hemidesmosomes
Stem cells often reside on the bottom layer connected to the basement membrane
c. No blood vessels (avascular), nutrients and waste transported by diffusion
Epithelium Functions
a. Act as a protective covering over tissue
b. Regulate transport through tight junctions and channels/transporters/pumps
c. Absorption from extracellular environments (i.e. using channels, transporters and pumps inserted in apical and basolateral membranes)
d. Secretion (Glands) - exocrine, paracrine, endocrine
Tight junctions regulate transport by:
Claudin/Occludin proteins
- paracellularly (between epithelial cells) transport
Channels/ Transporters/ Pumps regulate transport:
transcellular (across cell) transport
Epithelium Origin
All 3 germ layers (Endoderm, Mesoderm, Ectoderm)
Exocrine Glands
- secrete to the outside ducts and free surfaces (apical secretion)
Paracrine Glands
- secretes to underlying connective tissue (basal secretion) that produces a local effect
Endocrine Glands
- secretes to underlying connective tissue (basal secretion) and into the bloodstream where secreted product travels to, and acts at, distant sites (basal)
Apical and basal polarity is set up by:
- basal membrane attachment and cell -cell junctions
Terminal Bars are formed by:
- Adherens and tight junctions toward the apical side
i. polarity protein complexes associate with terminal bars to direct vesicle traffic apically or basolaterally depending on sorting sequences
ii. Actin filaments at terminal bars form a zonular “belt” apically all the way around the circumference of the cell
iii. Myosin can contract the actin belt to constrict the cells apically; Apical constriction the decreases the apical cell diameter while increasing basal cell diameter; collectively this ‘bends’ the epithelium and helps generate the formation of tubes (=morphogenesis)
Microvilli
i. Very small 1um long 25nm wide projections on apical surface. Viewable by E.M. but not individually by
L.M.; collectively can be seen as a ‘brush border’ on the apical aspect of the epithelium by L.M.
ii. Actin filament core that extends into cell and connects together at terminal web ( anchored to zonula
belt)
iii. Increases surface area for absorption (prominent in the small intestine for absorption of the products of
digestion)
iv. Not motile
Cilia
i. Larger; 10um long, 250nm wide projections on apical surface. Individual cilia viewable by both E.M. and
L.M.
ii. Microtubule core linked together by radial spokes (9,2 arrangement) action of microtubular motors
actively bends cilia = motility
iii. Collective ciliary motility produces a beating pattern across the top of epithelia to move material across
it (prominent in the respiratory system for debris clearance)
Simple Epithelia
1 layer, all cells are attached to basement membrane
Stratified Epithelia
More than 1 layer not all cells are attached to basement membrane
Pseudostratified Epithelia
Appears to be many layers but all cells are attached to basement membrane
Squamous Epithelia
Flattened cells
Cuboidal Epithelia
Cube-shaped cells
Columnar Epithelia
Taller than wide-shaped cells
Transitional Epithelia
apical cells can change shape from pillow-like to squamous depending state
of stretch/distension of the epithelium
Simple squamous
- Lining epithelia at locations of gas exchange (eg. inner blood vessel lining, body cavity lining, loops of henle, alveoli blood air barrier
Simple cuboidal
- Secretion, absorption, protection (eg. Ducts and kidney tubules)
Simple Columnar
- Transportation, absorption, secretory. Often have microvilli (eg. digestive and urinary
tracks)
Stratified squamous keratinized epithelia
- protective and waterproof, apical most layers consist of enucleated/dead cells that contain large amounts of keratin (= squames; found in the epidermis of the
skin)
Stratified squamous non-keratinized epithelium
protective, found in wet areas of transition between external and external transition of the body (eg. GI, Respiratory and Genitourinary tracts).
Pseudostratified epithelium
Multifunctional; cells are of different heights from cuboidal to tall columnar; appears multilayered due to
nuclei at multiple levels by L.M. but all cells attach to the basement membrane; thus, actually a modified simple epithelium; often ciliated and is prominent in the respiratory system
Transitional Epithelium
Truly stratified and distensible/stretchable; apical-most cells change shape as the epithelium becomes distended due to dynamic cell-cell junctions and pleating/folding of plasmamembrane between junctions; prominent in bladder and ureter which are often distended/stretched as they fill
Reticular Fibers of Connective Tissue
-very thin specialized form of collagen (Collagen Type III) fibers that are cross-linked together to form a meshwork/net within highly cellular connective tissues (eg. within lymphoid organs)
connective tissue cells can be generated from:
- pleuripotent mesenchymal stem cells (in the connective tissues) or pleuripotent hemopoietic stem cells (in the bone marrow; cells then travel to connective tissues through the bloodstream)
6 major cell types of connective tissue
Fibroblasts, adipocytes, pericytes, mast cells, macrophages, plasma cells
Connective Tissue Proper Classification
- Loose/Areolar CT
- Dense CT - irregular and regular
- Adipose CT
- Reticular CT
Loose/Areoler CT
many cells, abundant ground substance; few fibers; flexible not resistant to stress
Dense CT
few cells/more fibers; resistant to stress
Irregular Dense CT
fibers/strength in all orientations directions
Regular Dense CT
fibers in parallel arrays, strength in one direction
Adipose CT
high number of adipocytes grouped into lobules separated by dense irregular CT ‘septae’
Reticular CT
highly cellular connective tissue with interwoven reticular fibers
GLANDS
- epithelial portion is secretory (= parenchyma)
- connective tissue portion is inductive and supportive (= stroma)
- exocrine glands, secretory product is released apically into a duct or onto a free surface
- endocrine glands, secretory product is release basally into stroma, picked up bloodstream (=
‘hormone); factors that act locally on nearby cells after diffusing within the same tissue are called ‘paracrine’ factors
Types of Exocrine Glands
- unicellular glands = single cells embedded in a surface/lining epithelium (eg. goblet cells)
- multicellular glands = clusters of secretory parenchymal cells surrounded by stroma; secrete into a duct (eg. salivary glands)
Multicellular Exocrine Gland Classification
- duct morphology (simple = unbranched ducts; compound =branched ducts)
- secretory portion morphology (tubular=same diameter as duct; acinar/alveolar=expanded diameter
compared to duct) - nature of secretion (serous = proteinaceous, enzyme rich; mucous = proteoglycan, lubicricating)
Cartilage Functions
- supports soft tissues
- provides a low friction sliding area for joints
- promotes growth of long bones as a reserve of new bone deposition at the ‘growth plates’ of
endochondral bones
Cartilage Cells
- Chondroblasts start to deposit cartilage ECM in the outer portion of the tissue, arise from chondrogenic ‘chondroprogenitor’ cells located in the outermost ‘perichondrium’ connective tissue layer
- Chondrocytes are terminally differentiated from chondroblasts and are embedded in lacunae (small spaces) located deep within the tissue
Cartilage matrix
- Generally, water>collagen>proteoglycans; resilient/plastic/deformable, but resists compression
- Intermediate thickness Type II Collagen fibers provides limited tensile strength
- Avascular; therefore high diffusion rates through hydrated matrix is critical for cell survival
3 types of cartilage
(1) Hyaline cartilage (HC)
(2) Elastic cartilage (EC)
(3) Fibrocartilage (FC)
Hyaline cartilage (HC)
- Type II collagen fibers predominate
- 60-70% water, recruited by significant amounts of proteoglycans (PG) - Well-defined perichondrium, with limited regenerative potential
- Found in joints, nose and trachea (most prevalent type of cartilage)
Elastic cartilage (EC)
- Decreased matrix/increased number of cells compared to hyaline cartilage - Type II collagen + Elastic fibers present in the matrix
- Well-defined perichondrium, with limited regenerative potential.
- Found in very flexible areas (eg. outer ear)
Fibrocartilage (FC)
- Matrix contains high amounts of thick, strong Type I collagen fibers = very high tensile
strength compared to other type of cartilage - Also has a number of characteristics of dense regular connective tissue with some
chondrocytes in lacunae and some proteoglycans’s that give some resistance to
compression - No distinct perichondrium = very, very low regenerative potential
- Found in intervertebral disks, pubic symphysis, and sites of tendon insertion/attachment
to bones, cushioning meniscus in some joints (eg. knee cartilage that is often ‘torn’)
Bone Cells
- Osteoblasts: deposit osteoid (organic ECM), arise from osteogenic ‘osteoprogenitor’ cells in outer ‘periosteum’ connective tissue (note: some osteogenic cells can migrate in, with blood vessels, to the inner surfaces of bone that face the central marrow cavity (= ‘endosteum’)
- Osteocytes: fully differentiated from osteoblasts trapped within lacunae deep in the mineralized/rigid bone matrix
- Osteoclasts: resorb/breakdown bone matrix; multinucleate and derived from ‘hematopoietic stem cells’; Understand how ostoclasts breakdown of both the inorganic and organic portions of matrix (regulated by hormones from parathyroid and thyroid glands
Bone ECM (analogous to ‘reinforced’ concrete)
- Initially laid down by osteoblasts.
- Organic (‘osteoid’): high in type Type I Collagen = strong thick fibers= the ‘rebar’ reinforcement of the
bone ECM - Inorganic (mineralized’): contains hydroxyapatite (calcium phosphate crystals) for rigidity = the ‘concrete’ portion of the bone ECM
Structure of Bone Organs
- Periosteum covers external surface of bone; contains osteogenic cells with considerable regenerative potential;
- Inner Spongy Bone
- Outer Compact/Cortical Bone
Inner Spongy Bone
made up of spicules/small spikes (also known as ‘trabelulae) that are in contact with the bone marrow/; remodelled by ‘pitting’ by osteoclasts; pits/depressions/shallow lacunae; lacunae can subsequently be filled in by osteoid deposited by osteoblasts.
Outer Compact/Cortical Bone
- structural units (=’osteons’) formed by concentric layers of dense, bone tissue that form a cylinder with a central ‘Haversian’ canal
o Harversian canals contain blood vessels that run the length of the osteon
o Volkmann’s canals are tunnels that run perpendicular to, and connect, the Haversion canals between different osteons (see ppt slide and Fig 7-10 of textbook); both have blood vessels
(and nerves) running through them
o Note: individual osteocytes in the bone tissue form very small canals/tunnels (=’canaliculi’)
that contain long, extended cell process; these canaliculi connect from osteocyte to osteocyte
via gap/communicating junctions
Bone Formation (two types)
- Intramembranous
- Endochondral
Intramembranous Bone Formation
- generates flat bones, most prevalent in skull: forms directly in mesenchyme, which condenses and generates osteoprogenitor cells which then differentiate and lay down bone matrix
Endochondral Bone Formation
- generates most bones in body - forms within a pre-formed cartilage scaffold; ‘ossification centers’ form in which bone replaces cartilage within the scaffold; the central ossification center is the ‘diaphysis’, the outer ossification centers are the epiphyses; throughout childhood and puberty a zone of reserve cartilage (= ‘epiphyseal plate’ or ‘growth plate’) remains; the growth plate can proliferate (ie. increase in size) and then gradually ossify/become bone; the growth plate proliferates in response to growth hormone from the pituitary.
Osteoarthritis
- degenerative loss of articular hyaline cartilage, cannot be regenerated as cartilage leads to bone coming into contact with bone = painful and loss of joint stability (common with age and ‘wear and tear’; very different from rheumatoid arthritis which is caused by chronic inflammation within the joint which leads to cartilage destruction secondarily.
Osteoporosis
- bone homeostasis shifts towards resporption (loss) rather than deposition (gain) of bone ECM; this is prevalent in post-menopausal women as estrogen stimulates deposition; often treated pharmacologically by inhibiting the activity of osteoclasts.
General Points on Muscle Cells and Tissues:
• Mesodermally-derived.
• Can be striated (eg. striped due to sarcomeres; visible by L.M.) or non-striated (eg. no fully
organized sarcomeres, but still contractile).
• There are two types of striated muscle (eg. skeletal and cardiac muscle).
• There is one type of non-striated muscle (eg. smooth muscle).
• Muscle contraction can be voluntary or involuntary.
• Functions: locomotion/movement, maintenance of posture, respiration, constriction of
viscera (eg. rhythmic contraction = ‘peristalsis’ in GI and Genitourinary tracts), constriction of blood vessels, heart beat; to do so they must be contractile (=shorten) which is actin- myosin based.
• Note: in muscle, many structures are prefaced with the term ‘sarco’; thus sarcoplasmic reticulum = endplasmic reticulum in muscle cells
Skeletal Muscles
• Striated, voluntary.
• Myofibers (= skeletal muscle cells) are multinucleate (with nuclei at the periphery) and
contain myofbrils organized into myofibrils; cells are bundled together as groups =
fascicles.
• Individual myofibers attach directly to a surrounding basal lamina ECM which is similar to
the basal lamina that epithelial cells attach to; muscle cell attachment to the basal lamina is cell-ECM junction based and is mediated by the dystroglycan complex (see Ch 4, pg 83 and Lecture 13).
• Layers of connective tissue then surround the muscle: endomysium = fine sheath of reticular that surrounds individual myofibers; perimysium = dense irregular (collagenous) connective tissue that surrounds fascicles; epimysium = dense irregular (collagenous) connective tissue that surrounds the entire muscle . These connective tissue investments hold the muscle together during cycles of contraction and relaxation.
• Ends of muscle fibers attach to dense regular collagen fibers of tendons via finger-like extensions; this attachment is mediated by cell-ECM junctions where the adhesion molecules are integrins.
• The cells of skeletal muscle are the myofibers, which are multi-nucleated long, cylindrical and striated with nuclei located at the periphery/edges of the cell
Subcellular components of individual myofibers:
- Plasmamembrane = sarcolemma
- Cytoplasm = sarcoplasm
- Well-developed endoplasmic reticulum = sarcoplasmic reticulum
- Parallel arrays of thin actin and thick myosin myofilaments that are bundled in parallel arrays = a myofibril (note: there are many myofibrils within a single skeletal myofiber)
- Each myofibril is surrounded by mitochondria, sarcoplasmic reticulum (muscle ER), the terminal cisternae (= expansions) of the sarcoplasmic reticulum, and deep invaginations of the sarcolemma that are perpendicular to the length of the myofiber (=T tubule) (see Fig 8.5). Two terminal cisternae of the sarcoplasmic reticulum and a single T-tubule come very close together to form a triad (see more below).
Sarcoplasmic reticulum (SR)
- Stores intracellular calcium (when the muscle is at rest); releases calcium into the sarcoplasm when the muscle is stimulated by motor neuron firing.
- The SR is a modified smooth ER (eg. devoid of ribosomes) that forms an interconnected network of tubules.
- Two expanded sacs of the SR (= ‘terminal cisternae’) are very closely opposed to deep invaginations of the sarcolemma (=T-tubules) to form a triad. Therefore, the ‘triad’ is actually a specialized ‘ER junction’.
- A membrane depolarization/impulse generated by motor neurons that interact with the myofiber travels along the sarcolemma into the T-tubule…This stimulates the terminal cisternae to release calcium into the sarcoplasm very near the myofibrils….This stimulates the contraction of the sarcomere.
Sarcomere = The Contractile Unit of the Myofibrils
• One sarcomere is defined as the small region of a myofibril between two successive Z discs where thin actin filaments insert (see also text page 160 for description of A-band, M-line and I-band).
• The sarcomere contains thick (myosin) and thin filaments (actin).
Myosin: coiled-coil myofilament composed of two heavy chains, each with a globular head at the N-terminus, and four light chains.
Actin thin myofilaments: has tropomyosin, actin, and troponin complexes, which bind calcium.
• The thin filaments originate/are physically linked to proteins in the Z disk and project toward the center of the sarcomere (see Fig 8.8).
• The thick filaments form parallel arrays that cross the center of the sarcomere and partially overlap with the thin filaments.
• In the relaxed state, the thin filaments do not reach the midline and the thick filaments do not extend the entire length of the sarcomere (ie. they don’t reach/attach to the Z disk)
• During contraction, individual thick and thin filaments do not shorten. Instead, the two Z disks are brought closer together as the thin filaments slide past/are pulled over the thick filaments towards the center of the sarcomere (eg. Huxley’s sliding filament theory, see pg 162 of textbook).
Calcium mediated muscle contraction and relaxation steps:
- binding myosin head to actin
- Skeletal Muscle Contraction
Binding of the myosin head to actin
- Upon stimulation, the sarcoplasmic reticulum releases calcium into the sarcoplasm.
Calcium binds - Tropnin binds cacium which changes its conformation/shape
- Tropomyosin, which is attached to troponin, changes its position on the actin
filaments. This shift unmasks the myosin-binding sites on the actin. - Myosin heads can now bind actin filaments.
Skeletal muscle contraction:
- ATP hydrolysis (ATP is split to ADP and inorganic phosphate ‘Pi’) causes the cocking
of the myosin head to occur, and the myosin head is in its high-energy configuration. - Myosin attaches to the actin myofilament (in a calcium-dependent manner as
described above). - As ADP and Pi are released, the myosin head bends as it pulls on the actin filament,
dragging it to the center of the sarcomere. - As a new ATP attaches to the myosin head (low-energy configuration), the myosin
head detaches from actin. - As a result the actin is pulled by the myosin towards the center of the sarcomere (ie.
the thin actin filaments ‘slide’ over the thick myosin filaments
Sliding filament model of contraction:
When a muscle is relaxed, actin and myosin filaments overlap slightly.
When a muscle if fully contracted, actin filaments slide along the myosin, resulting in a great overlap and shortening of the sarcomere (ie. the Z-discs/lines come closer together = sarcomere and myofibril shortening
Somatic/Voluntary Motor Neuron Innervation
- The very specific immunostaining shows the axons (green; neurofilaments); blue (myelin sheath covering the nerve that ends before the axon terminal is reached) and the acetylcholine receptors in the sarcolemma/plasmamembrane of the end plate.
- Upon depolarization due to an action potential traveling down the neuron, pre-synaptic vesicles containing the neurotransmitter acetylcholine (ACh) fuse with the axon terminal plasmamembrane in a SNARE-dependent manner. As a result, ACh is released into the synaptic cleft (= extracellular) where it binds to ACh receptors on the sarcolemma and triggers a depolarizing action potential that travels down the T-tubule; thisinduces Ca2+ release from the terminal cisteranae of the sarcoplasmic reticulum in the triad; the result is a contraction of the sarcomeres as described in Lecture 12. This process is known as T- tubule-based ‘Excitation-Contraction Coupling’.
Botox:
Is a multimeric toxin produced by the bacterium Clostridium botulinum that prevents the release of Ach from axon terminals by inhibiting the fusion of ACh-containing vesicles with the pre-synaptic axonal membrane at the neuromuscular junction (given that, you can probably deduce one of the molecular targets of the toxin - or you can check Wiki). Large amounts of botox leads to botulism that causes muscular paralysis, respiratory failure, and even death, because muscle contraction cannot occur. It is used cosmetically in much smaller doses to paralyze muscle contraction to decrease age-related ‘wrinkling’
Duchenne Muscular Dystrophy (DMD):
• Loss of function mutations in the scaffold/adapter protein ‘dystrophin’ causes the cell-ECM junction ‘dystroglycan complex’ to be released from the actin cytskeleton. This results in contraction-mediated damage/tearing of skeletal myofibrils. Ultimately, this leads myofiber cell death which leads to increased connective tissue deposition (=fibrosis/scarring and adipocyte) proliferation as well as inflammation within the muscle.
- At the gross anatomical level this results in a swelling of the muscle = pseudo-hypertrophy. • Attempts to correct the defect are being made using viral-mediated transfer of the wild-
type dystrophin gene into myofibers.
Cardiac Muscle
• Found exclusively in the heart
• Cardiac muscle cells = cardiac myofibers = cardiomyocytes form the ‘myocardium’ (ie. the
thick contractile walls of the heart’s ventricles and the atria).
• Cardiac myofibers are striated with true sarcomeric organization in myofibrils, but they are
mononuclear (= one central nucleus per fiber) and their contraction is involuntarily
controlled (fibers contract spontaneously with inherent rhythmicity).
• Cardiac myofibers are short and branched and they attach to each other by intercalated
disks
• T tubules are present that have sparse amounts of sarcoplasmic reticulum associated with them (one tubule/one cistern = diad) that functions to release small amounts of calcium into the sarcoplasm upon depolarization. In addition, channels in the T-tubule membrane facilitate the passage of large amounts of extracellular calcium into the sarcoplasm to facilitate contraction after depolarization (see textbook pg 177/8)
intercalated disks contain:
1) Transversely-located (across the fibers at right angles) adherens junctions and desmsomes which are adhesive (ie. cardiomyocytes are attached to each other, usually at their branched ends (cardiomyocyes do not have myotendinous insertions like skeletal muscle myofibers as they do not attach to bone)
2) Laterally-located (parallel to the myofilaments) gap junctions which electrically couple
the cardiac myofibers by allowing for rapid flow of information between fibers. This is important for the inherent rhythmicity of cardiomyocytes as it means that the stimulation
of one cell can lead to the stimulation of neighbouring cells via gap junction transfer of ions).
Smooth Muscle
• Found in the walls of tube-based organs/structures where their contraction decreases the diameter of the central lumen (eg. during rhythmic, peristaltic contractions in the gastrointestinal and urogenital systems; to decrease blood flow through small arteries and arterioles)
• Smooth muscle myofibers are spindle-shaped and tapered each end, and they are mononucleated(= one nucleus per fiber).
• Contraction is involuntary and is initiated by multiple contacts from the axons of neurons from the ‘autonomic’ nervous system ); there are no well-defined neuromuscular junctions.
• Have no T-tubule-based excitation-contraction coupling; large numbers of gap junctions between smooth muscle myofibers allow direct electrical communication between adjacent smooth muscle fibers. This direct electrical communication facilitate slow rhythmic contractions across smooth muscle tissue.
• There are no sarcomeres; therefore smooth muscle is not striated.
• Cells have an extensive array of interweaving thin (actin) and thick (myosin) filaments that
give some multi-directionality to the contraction (ie. they are arranged at oblique angles rather than in parallel arrays as is the case in skeletal and cardiac myofiber sarcomeres); thin filaments attach to aggregates of adapter/scaffold proteins called dense bodies that include actin bundling proteins (eg. alpha-actinin) and act as an anchor for the filaments (analagous to Z-lines in skeletal/cardiac muscle)
• Contractile units consist of myosin filaments connecting to the actin filaments:
Contractile units consist of myosin filaments connecting to the actin filaments:
• Thick filaments (myosin) act on thin filaments to cause contractions between the
dense bodies and dense plaques; this leads to shortening of the long, tapered, fusiform-shaped cells as well as some cell twisting. In histological sections this contractile twisting causes the nuclei to take on a ‘corkscrew’-like appearance.
Examples of sites of smooth muscle:
• Arteries and veins: in some of these blood vessels they are found in the central tunica
media (see Lect 19 for more specific details)
• Multiple layers in the wall of the stomach and intestines: that act together to
generate peristalsis (= progressive and rhythmic contractions of the smooth muscle that propel food through the digestive tract); See Lect 27.
Overview of Nerves:
• The neuron is the functional cell of the nervous system that transmits this information. Therefore neurons have unique features that allow them to perform this function.
• Nervous tissue develops from ectoderm. Some of the general functions of nervous tissue are: to detect and analyze sensory input; coordinate body activities, store (= learning) and recall (= memory) experiences
• There are two parts of the nervous system:
Central Nervous System (CNS)
Peripheral Nervous System (PNS)
• There is bi-directional information flow between the CNS and PNS.
Central Nervous System:
• Consists of brain and spinal cord
• Organized ‘somatotopically’ in that different brain areas are dedicated to
serving/responding to different body parts and/or different modes or types of information
• Certain body parts with fine motor and sensation capabilities (ie. oral cavity and hands) have a greater somatatopic representation in the brain (ie. there is more neuronal tissue = larger portion of the somatatopic ‘map’ that regulates or receives information from them).
• Spinal cord: white matter contains tracts of myelinated axons where the information is ascending (ie. sensory information going up to the brain) or descending (motor information leaving the brain).
Gyri
“bumps” on the cortical portion of the brain
Sulci
“grooves” on the cortical portion of the brain
Primary Motor Cortex
the precentral gyrus, which is located just anterior to the large central sulcus of the brain (where voluntary motor information originates in the brain)
Primary Sensory Cortex
the postcentral gyrus, which is the gyrus posterior to the central sulcus (where conscious sensory information first arrives in the brain)
Grey matter
neuronal cell bodies, dendrites, unmyelinated axons
White matter
tracts of myelinated nerve axons/fibers
Peripheral Nervous System
• = All nerves of the body that are outside the CNS, often mixed motor and sensory nerves
• Sensory component of peripheral nerves carry information from the body to the CNS =
afferent nerves
• Sensory/afferent fibers enter the spinal cord via dorsal nerve roots
• Motor component of peripheral nerves carry information from the CNS to the body to
effector organs (like muscles and glands) = efferent nerves
• Motor/efferent fibers exit the spinal cord via ventral nerve roots (which was originally the
‘floor plate’ in the developing neural tube; see Lect03)
Somatic Portion of the PNS
- somatic sensory nerves carry information that is consciously perceived.
- somatic motor nerves carry information that leads to voluntary skeletal muscle contraction
- consists of single neuron connection
Autonomic/Visceral Portion of PNS
-Sensory nerves receive unconscious ‘propioceptive’ (ie. regarding position of body in space) information
-Motor nerves carry information that leads to the contraction of involuntary smooth and/or cardiac muscle, as well as the stimulation of the secretion of a number of glands
- consists multiple neuron connections (synapses occur in peripheral autonomic ‘ganglia’) -The autonomic motor system further broken down into the sympathetic division (1st/preganglionic neurons originate in the thoracic and lumbar regions of the spinal
cord; for ‘flight/fright’ in catabolic responses) and the parasympathetic division (1st/preganglionic neurons originate in the cranial, cervical & sacral regions of spinal cord; for ‘rest/repose’ in anabolic responses)
3 Categories of Nervous Tissue
1) Neurons
2) Neuroglial Cells
3) Support Cells
Neurons
from ectoderm/neuroectoderm
- excitable cells that carry out information transfer
Neuroglial Cells
from ectoderm/neuroectoderm
- non-excitable cells that enhance
efficiency of transmission
Support Cells
from mesoderm
- include cells of blood vessels (endothelium, fibroblasts) and microglia (immune cells of the CNS)
Neurons Consist of:
- soma
- Dendrites
- axon
- Axon Terminal
Soma
cell body/perikaryon; has a nucleus with prominent nucleolus; the cytoplasm has cytoskeleton, organelles, Golgi apparatus, mitochondria, and ribosome-rich RER that stains with hematoxylin and, especially with Nissl stain (both are histochemical; bind acids)
Dendrites
receptive region of the cell = conduct impulses towards the cell body; has similar contents as the soma, with mitochondria, smooth ER, microtubules (move organelles and vesicles) and neurofilaments (structural backbone)
Axon
conductive region of the cell = conducts nervous impulses away from cell body towards axon terminal; has mitochondria and SER (but few/no ribosomes or RER; no protein synthesis), has many microtubules for the transport of vesicles and organelles up and down the axon
Axon Terminal
‘end bulb’; portion of the axon that comes into close proximity with a target cell (another neuron, or muscle cell, or glandular cell) at a connecting ‘synapse’ where information is passed
Synapses
- Site of neuron to neuron interactions (eg. axodendritic and axosomatic depending on location)
- Modified at sites of neuron-to-muscle cell (i.e. the neuromuscular junction; see Lect 13) and neuron-to-gland cell interaction.
Synapses Consist of:
- presynaptic axon Terminal
- synaptic Cleft
- post Synaptic terminal
Presynaptic Axon Terminal
site of vesicle accumulation that, in response to a conductive
action potential that travels down the axon, fuse with the plasma membrane (via SNARES)
to release neurotransmitter by exocytosis into the…
Synaptic cleft
intercellular/extracellular space between the two neurons that contains the
released neurotransmitter during ‘synaptic transmission’ such that it can bind to the…
Post-synaptic terminal
which has cell surface receptors to receive neurotransmitters that,
when bound, initiate a receptive action potential in the neuron that receives the signal.
