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• Introduction to CTB
• Basic Cellular Structures
• Epithelial Tissue
• The Cell Cycle
• Muscle Tissue
• Connective Tissue

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DYES:

• Dye Structure:
• Chromophore Group: The chemical moiety of the dye that is responsible for its color.
• Auxochrome Group: The moiety on the dye that binds to the cellular components. It is usually either amino or SO₄^2- groups.
• Amino auxochrome group = a basic dye.
• Sulfate auxochrome group = an acidic dye.
• Common types of stains:
• Hematoxylin and Eosin (H&E): Most common type of stain.
• Hematoxylin: Functionally a basic dye (despite the fact that it is anionic). It binds to basophilic (negatively charged) nuclear components like DNA and RNA.
• It stains blue
• Eosin: Acidic dye. It binds to positively charged, acidophilic components.
• It stains pink to red.
• Masson (Trichrome) Stain:
• Collagen is green.
• Elastic fibers are red.

ACIDOPHILIC: Attracted to acidic substances, which are anionic (negatively charged) at physiologic pH. Thus acidophilic substances are positively charged.

• Proteins are acidophilic in at a pH higher (more basic) than their isoelectric point. When the environmental pH is above a protein's isoelectric point, the protein is positively charged and hence acidophilic.
• Many proteins are acidophilic at physiologic pH.
• Acidophilic Components:

BASOPHILIC: Attracted to basic substances, which are cationic (positively charged) at physiologic pH. Thus basophilic substances are negatively charged.

• Proteins are basophilic at a pH lower (more acidic) than their isoelectric point. When the environmental pH is below a protein's isoelectric point, the protein is negatively charged and hence basophilic.
• Basophilic Components:
• DNA and RNA = basophilic due to presence of phosphate groups.
• Proteoglycans = basophilic due to sugars and esterified sulfates which are negative at physiologic pH.

Special Types of Staining Techniques:

• Metachromasia: A substance can take on a different than expected color when the substance has two chemically reactive groups that interact due to their close proximity.
• Fat-Staining: To stain membranes and lipid-materials, you must use a fat-insoluble solvent and freeze-fracturing. You can't use paraffin because it would dissolve the substance!
• Common solvents include propylene glycol, and ethanol.
• Sudan IV is a typical fat-soluble dye.
• The Schiff Reagent -- specific for DNA and polysaccharides.
• Feulgen Reaction: This reaction uses Leucofuchsin as a dye, which selectively stains purines in DNA.
• Periodic Acid-Schiff (PAS) Reaction: Selectively stains polyhexoses and hexosamines. Tissues stained by this reaction include:
• Glycogen
• Epithelial mucins in goblet cells.
• Proteoglycans in basement membranes -- but not of the CT matrix.
• Enzymatic Staining: For example, you can visualize mitochondria by testing for the product of a mitochondrial enzyme. The important point is that the enzyme is not stained directly in these procedures. Rather, the localization of its activity is tested for.
• Immunohistochemistry:
• Fluorescent Antibody Technique: Complex a fluorescent dye with an antibody that binds to specific antigens on tissues that you want to visualize.
• Indirect Immunofluorescence: Visualization of a tissue using two antibodies, where the target structure that is actually visualized is bound to the second antibody.
• Indirect Immunocytochemistry: Similar to indirect immunofluorescence, but eliminating the need for fluorescent visualization.
• Protein-A Gold Technique:
• Autoradiography: beta-electrons interacting with silver bromide (AgBr) crystals from radioactive materials illuminates radioactive structures.
• Electron Microscopy:
• Staining is usually with osmium.
• Some sort of fixation is required -- such as Freeze Fracture, in which we cut a preparation into thin slices using a microtome.

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FLUID MOSAIC MODEL:

RED-BLOOD CELLS GHOSTS: Put a RBC in salt and crack the membrane (i.e. make it leaky) so that all contents leak out. Then reseal the membrane, and we are left with topography maps of the RBC-membrane, showing peripheral and integral membrane-proteins.

• Integral Proteins:
• Glycophorin: Has extensive saccharide groups on the exterior surface.
• It is a single-pass protein.
• Band-III: Peripheral anion channel, exchanging HCO₃^- out for Cl^- in.
• It is a multi-pass integral membrane protein.
• Band-III has no lateral mobility in the membrane -- it is hooked directly to the cytoskeleton via Ankyrin ------> Spectrin ------> Actin spokes.
• Rhodopsin: The "mother" of the 7-pass alpha-helical multi-pass transmembrane protein (of the adrenergic G-protein-bound receptor family). This is a general class of integral proteins and describes a lot of different proteins.
• Peripheral Proteins
• Ankyrin is connected to the inside periphery of the RBC membrane.
• Spectrin is hooked to membrane via Ankyrin.
• Spectrin forms a lattice network composed of alpha and beta dimers.
• It hooks onto Band-III in the membrane (via ankyrin) at one end, and onto Actin at the spokes of the RBC-cytoskeleton in the RBC interior.
• Band 4.1: Another peripheral protein that helps anchor spectrin and actin to the RBC membrane.
• Hereditary Spherocytosis: Hemolytic anemia caused by a failure for RBC's to form a biconcave disc and therefore inability to squeeze through capillaries.
• It can be caused by any of a number of genetic mutations in RBC cytoskeletal proteins.
• One form is caused by a mutation in Ankyrin which results in bad splicing. There is a 2.1 and 2.2 splice out of the same precursor mRNA.
• 2.1 splice: predominant in developing cells.
• 2.2 splice required in mature cells.
• The 2.2 splice disappears with the missplicing mutation, hence RBC's mature but they don't function when fully developed.
• At the same time, other ankyrin isoforms of the same RNA precursor are translated normally, but they are in other cell-types.

GLYCOSYLATION:

• N-Linked Glycosylation
• Sugar hooks onto Asparagine Residue.
• Common Sugars attached are N-Acetylglucosamine (GluNAc), and Mannose
• Glycosylation occurs cotranslationally, in the Rough ER.
• PROCESS:
• Core Glycosylation event occurs initially. It involves the linkage of the core oligosaccharide.
• The core oligosaccharide is then associated to the lipid complex, dolichol phosphate. Then it is disassociated and linked to the protein in one step.
• O-Linked Glycosylation
• Sugars hook onto Serine or Threonine residues.
• Common sugars attached are N-Acetyl Neuraminic (Sialic) Acid and N-Acetylgalactosamine.
• Glycosylation occurs posttranslationally, in the Golgi.

Experiments to Demonstrate the Fluid-Mosaic Model: Lipids can move laterally and can wiggle their hydrophobic tails very rapidly, but they can't flip-flop without a special catalytic reaction (catalyzed by flippase).

• Heterokaryon Experiment: Showed the movement of membrane proteins within the plasma membrane of a human-mouse hybrid.
• Fluorescence Recovery After Photobleaching (FRAP): A way to show that lateral movement of membrane proteins occurs.
• You can determine a Diffusion Coefficient for Lateral Mobility. Some common coefficients:
• Phospholipids in membranes: 1 x 10^-8 cm²/sec
• Most highly mobile membrane protein (Rhodopsin): 5 x 10^-9 cm²/sec
• You start with 100% fluorescence in membrane, then zap with bleach a little spot on the membrane, and the fluorescence goes way down to about zero.
• Then you can watch the fluorescence recover (back up to near 100%) as adjacent lipids and/or proteins diffuse to the bleached area.

Restricted Mobility: The cytoskeleton in red blood cells restricts the mobility of many membrane proteins on the RBC membrane.

Cytoskeletal Elements:

Filament Type	Size	Composition
Microfilaments	7-8 nm	Actin monomers
Intermediate Filaments	10 nm	variable
Microtubules	25 nm	alpha and beta tubulin monomers
Myosin (Thick) Filaments	variable	Myosin

Microtubules:

• Made of dimers of alpha and beta tubulin. They will self-assemble (autopolymerize) under the right conditions.
• Polarity
• (+)-End: Tubulin monomers are, on average, being added to this end. New monomers are put on at a faster rate than they fall off.
• (-)-End: Tubulin monomers are, on average, being removed from this end. Monomers fall off at a faster rate than they are put on.
• Microtubule Organizing Center (MTOC): Often found around centrioles. Microtubules hook to centrioles by their (-)-ends.
• Tread milling Effect: If you label one monomer on a microtubule, it will appear as if it magically moves from the plus to the minus end.
• That's because we keep adding new monomers to the plus end, so it gets pushed further back in the chain, until finally it is all the way toward the minus end and it falls off the chain.
• Anti-Microtubule Drugs:
• Colchicine: Binds to tubulin monomers and thereby prevents assembly of microtubules, killing the cell.
• Taxol: Controversial new anti-cancer drug that works in the exact opposite way as traditional drugs. It stabilizes the microtubule filament so that it can't disassemble. The result is the same, however: microtubule dynamics are lost and the cell dies.

CYTOSKELETAL MOTOR PROTEINS: ATPases that cleave ATP to cause movement. The microtubules / actin don't move themselves. Rather it is the interaction of the motor proteins with the tubules that causes movement.

• Myosin: Actin-binding protein.
• Dynein: (-)-End Oriented Microtubule binding protein.
• It moves along the microtubules from the (+) to the (-) end. It therefore facilitates retrograde axonal transport.
• Tail is the region that attaches to the microtubules. The Head is the ATPase region.
• Kinesin: (+)-End Oriented Microtubule binding protein.
• It moves along the microtubules from the (-) to the (+) end. It therefore facilitates anterograde axonal transport.
• Cilia/Flagella: The minus end is toward the tip, and the (+)-end is toward the basal body, toward the plasma membrane.

INTERMEDIATE FILAMENTS: Made of keratins, desmin, vimentin, and neurofilaments.

NUCLEAR TARGETING of PROTEINS:

• Nuclear Pores: Have specific targeting signals for nucleus-bound proteins. Pores are formed at points where the inner and outer layers of the Nuclear bi-membrane come together.
• EXPT: The Large-T Antigen of the SP40 virus was seen in the nucleus of a host cell by immunocytochemical imaging.
• A mutation on the T-Antigen site, exchanging a Lysine for a Threonine, caused sorting to occur in the cytosol instead.
• Thus this mutation was part of the Nuclear-Targeting Sequence.
• EXPT: Frog oocytes -- the results suggested that the nuclear targeting sequence was on the tail subunit of the nucleoplasmin protein in frog oocytes.
• When the head and tail were dissociated, the tail was able to through nuclear membrane and head wasn't.
• Also, if colloidal gold particles are associated with this tail subunit, they, too, can get into the nucleus, but only if ATP is present.
• SUMMARY:
• Transport into the nucleus does not take place by passive diffusion. It takes by highly specific transport with targeting sequences.
• It appears that nuclear transport is an active process (at least in frog oocytes). It requires ATP.

ROUGH ENDOPLASMIC RETICULUM: Cytosolic proteins can be synthesized on free ribosomes instead of the Rough ER, per se. However, the following proteins are always synthesized on the Rough ER:

• Membrane Proteins: Using Signal Peptides and Signal Recognition Particles, they are directly translated into the membrane, where they stay.
• Secreted Proteins: They are exuded into the ER lumen, and then onto Golgi and finally secreted in vesicles. They must be synthesized on ER therefore.

MITOCHONDRIA: Proteins destined for the mitochondria are integrated into the mitochondrial membrane post-translationally. First they are synthesized, and then they go to mitochondria via a vesicle.

GOLGI COMPLEX:

• Cis Golgi: Earliest part of Golgi, closest to the ER.
• Transition Vesicles often transport material from the ER to the Golgi.
• Middle Golgi
• Trans Golgi: Part of Golgi off of which vesicle bud.

ENDOCYTOSIS: Clathrin associated with a receptor protein, which in turn associated with the membrane.

• There are several adapter proteins, depending on the membrane to which the vesicle will fuse. For example, there is a specific adapter protein for the Golgi.
• The difference in adapter proteins between

LYSOSOMAL STORAGE DISEASES: Lots of diseases have at least one etiology where the mutation lies in incorrect sorting of the protein, rather than a non-functional protein itself.

• I-Cell Disease: The Mannose-6-Phosphate recognition marker is found on one of the N-Linked Oligosaccharides of a lysosomal hydrolase. It targets the protein for the lysosome. Adding the M6P is a two step process.
• One enzyme puts on N-Acetylglucosamine phosphate onto a mannose residue.
• A second enzyme then removes the N-Acetylglucosamine, leaving Mannose-6-Phosphate in its wake.
• It is the first step, addition of N-Acetylglucosamine phosphate, that goes wrong in I-Cell disease.
• Cystic Fibrosis: The CFTR protein is mostly getting made, but it is not getting transported to the Golgi. The primary etiology of the disease is a sorting problem, not a defective protein.
• Tay-Sach's Disease: Again, one of the causes is a missorting of the protein beta-Hexosaminidase, where it can't get from ER to Golgi.
• Emphysema and Familial Hypercholesterolemia are two more examples.
• Sucrase-Isomaltase Deficiency:
• The Sucrase-Isomaltase enzyme is normally targeted to the apical epithelial membrane and is involved with disaccharide / glycogen breakdown.
• Individuals with the defect can't metabolize long-chain sugars.
• Again it seems that the secretory pathway for the enzyme is blocked.

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EPITHELIAL CELL TYPES:

• Simple Squamous Epithelium: Kidney Bowman's Capsule
• Resemble fried eggs in shape.
• Simple Cuboidal Epithelium: Kidney Collecting Tubule
• Kidney tubules cells are specialized for absorbing salt and water in an apical to basal direction.
• Simple Columnar Epithelium: GI Tract (Stomach, Jejunum, Duodenum, Ileum)
• Other Tissues: Gall Bladder and Uterine Gland.
• Simple Columnar Cells are specialized for one or all of three things:
• Secretion
• Protection
• Absorption: This is especially true in Duodenum and Jejunum.
• They have oval nuclei toward the basal side.
• SIMPLE COLUMNAR EPITHELIUM CELL TYPES: There are four basic cell types of simple columnar epithelia
• Columnar
• Fusiform
• Basal
• Goblet: = Modified columnar cells that synthesize and secrete mucous.
• Stereocilia are "cilia" that don't move, but they are actually very long microvilli specialized for absorption, and only visible at EM level.
• Pseudostratified Columnar Epithelium: Trachea and Upper Respiratory Tract
• The trachea is actually ciliated, but there are also non-ciliated pseudostratified columnar epithelia.
• Example of Pseudostratified Non-Ciliated Columnar Epithelium: Male Urethra
• Stratified Squamous Epithelium: Salivary Glands, Skin, Vaginal Wall
• There was no example of this in the carousels but only final testing slide.
• Stratified Squamous Keratinized: Layer of Keratin on top, as in Skin.
• Stratified Squamous Non-Keratinized (Mucosal): No Keratin on apical surface, as in Vagina and Mouth.
• Stratified cells form the following layers:
• Basal End: Cuboidal Cells that are proliferative.
• Middle: Polygonal cells held together by desmosomes.
• Apical End: Squamous Cells that are non-proliferative.
• Stratified Cuboidal Epithelium: Sweat Duct of Skin
• Transitional Epithelium: Urinary Bladder
• The tissue appears to transform from 5-8 layers when empty, to 2-4 layers when the bladder is filled. The cells can squish together.

EPITHELIAL General Characteristics

• AVASCULAR: Epithelial Tissue is generally avascular.
• POLARITY: Epithelial cell have polarity.
• The apical side often contains microvilli and faces the lumen of whatever surface the epithelium lines.
• Microvilli are characteristically found on apical domain. Actin filaments are associated with the microvilli, forming the terminal web.
• Cilia are found on apical membrane, in ciliated cells.
• The basal side is opposite that. A basement membrane, consisting of a basal lamina and reticular lamina, often underlies that.
• The Na⁺/K⁺-ATPase pump is characteristically only found on the basolateral membrane.
• BASEMENT MEMBRANE: The basal lamina is visible only at the EM level. The Basement Membrane, on the basal surface, is available at the LM level and consists of the basal lamina plus the underlying connective tissue.
• MESOTHELIUM: Mesodermally derived epithelium that lines body cavities.

TERMINAL WEB: Visible network of actin filament on the apical end of an epithelial cell.

JUNCTIONAL COMPLEX: The junctional complex keeps the apical and basal sides of the epithelium separate from each other.

• Zonula Occludens: Tight Junctions. They allow for selective passage of particles, and they prevent particles from getting stuck between cells or getting into the lumen.
• Zonula Adherens: Also present at the junctional complex.
• Macula Adherens: Desmosome. It goes all the way around the circumference of the cell, like a belt or a spotweld.
• TERMINAL BAR: Zonula Occludens + Zonula Adherens.
• Gap Junction: Believed to mediate electronic coupling between cells. Dye can squeeze through a gap junction to get one from cell to the neighbor.

POLARITY EXPT: Cells lost their polarity by disassociating and then reassociating cells such that they lose their intercellular contacts.

• The Na/K ATPase pump occurs only on the basal membrane of the cell.
• Viral EXPTs: You can also study the distribution of viral proteins to study the host-cell's machinery, since the virus uses the host-cell's machinery.
• People have watched where viral capsid proteins went when they associated with the host plasma membrane.
• The Influenza Virus only distributed proteins to the apical end of an epithelial cell.
• PATHWAYS for Explaining Polarity: Two alternative methods have been figured out.
• Targeting Mechanism where a class of vesicles specifically recognize proteins on the apical domain. Hence some proteins will only merge with membrane on the apical domain.
• Transcytosis: Some evidence also suggests that proteins are initially sorted in the basal domain, and then later transferred to the apical domain via transcytosis.

EPITHELIAL EXOCRINE GLANDS:

• Unicellular: Goblet Cells are unicellular exocrine glands.
• Simple Tubular
• Simple Branched Tubular
• Simple Alveolar
• Simple Branched Alveolar
• Compound Tubular
• Compound Alveolar
• Compound Branched Tubular
• Compound Branched Alveolar

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Types of Cells Cycles:

• Chromosomal Cycle
• Centrosomal Cycle: The Centrioles duplicate themselves prior to mitosis, and move to opposite poles.
• Cytoplasmic Cycle: Refers to cytokinesis. Distribution and redistribution of cytoplasm.
• Phosphorylation Cycle: Phosphorylation promotes mitosis, as discussed later.
• Nuclear Membrane Cycle: Nuclear Lamins are phosphorylated during Prophase, which causes them to dissociate and results in breakdown the nuclear membrane.
• Nuclear lamins are a form of intermediate filament.
• Nuclear Lamins are dephosphorylated during telophase, so they reassociate and membrane reforms.

CENTROSOMES: They divide into two before mitosis.

• They form the Microtubule Organizing Center, out of which the mitotic spindle grows, during mitosis.

MITOSIS:

• Prophase:
• Nucleoli disappear
• Centrosomes split and each daughter forms an aster.
• Prometaphase
• The Nuclear Envelope breaks down.
• Microtubules from each centrosome start interacting with the chromosomes.
• Kinetochore Microtubules from the centromere of each chromosome mature and attach to some of the spindle microtubules.
• Metaphase
• The Kinetochore microtubules align the chromosomes along the metaphase plate.
• The chromosomes are held in place by the opposed kinetochores and their associated microtubules.
• Anaphase
• Kinetochores on each chromosome separate, allowing each chromatid to be pulled toward the poles.
• Anaphase-A: Kinetochore Microtubules shorten. Since the plus end of these microtubules is right at the centromere, this shortening causes the chromosomes to be pulled toward the poles.
• Anaphase-B: Polar Microtubules elongate. The plus end of the polar microtubules face the equator too, but this elongation somehow aids in pulling (or pushing) the poles apart.
• Ca⁺² seems to play a role in promoting anaphase. There is high Ca⁺² concentration during anaphase.
• Telophase:
• Daughter chromatids reach the poles.
• Kinetochore microtubules disappear.
• Nuclear envelope reforms as nuclear lamins reassociate, condensed chromatin expands, and nucleoli reappear.
• Involves dephosphorylation of many proteins.
• Cytokinesis.
• Actin and Myosin pinch the cell and form a contractile ring.
• Organelles and cytoplasm are distributed evenly.

KINETOCHORES: Protein masses that form at the centromeres during mitosis, and to which kinetochore microtubules attach.

• SCLERODERMA: These patients produce auto-antibodies that react specifically with kinetochores.
• The Kinetochore Microtubules elongate toward the chromosome! They have their plus-end facing the chromosome, hence they shorten during chromosome separation.
• Both Kinetochores must be attached for the separation to occur. This is a biological safeguard to assure that nondisjunction does not occur.

CELL FUSION EXPERIMENTS: They provided evidence for activators that promoted mitosis and DNA Synthesis. Cells in different stages of the cell cycle were fused together to see what would happen.

• G₁ Cell + S Cell: G₁ Cell immediately goes into DNA-Synthesis.
• This is because the S-Cell had S-Phase Activator, which promoted theG₁ cell to go into S-Phase.
• G₁ Cell + G₂ Cell: G₁ will go through S-Phase as normal until it reaches G₂, then the two cells will go through mitosis together.
• So, the G₂ cell waits for the G₁ cell to catch up with it.
• This suggests that S-Phase Activator present in the S-Phase is no longer functional in the G₂ phase. This is important -- it prevents polyploidy by not allowing cells to synthesize DNA twice!
• G₂ Cell + S Cell: Again, S-Phase cell catches up to G₂ cell, then they proceed through mitosis together.
• This expt demonstrated that their was no S-Phase Inhibitor in the G₂ cell, or else the S-cell wouldn't have completed mitosis.
• Thus there must be some other explanation for why the G₂ cell doesn't undergo replication in presence of S-Phase Activator.
• Any Interphase Cell + M-Phase Cell: The interphase cell will prematurely enter mitosis, from any stage, resulting in an abnormal cell.
• This is mediated by M-Phase Promoting Factor (MPF), as below.
• DNA-DAMAGE: When G₂ cells are irradiated, their entry into M phase is delayed. They don't enter mitosis until their DNA-repair processes are complete!

CELIAC DISEASE: Intestinal disease results from abnormalities in intestinal epithelial cell division.

• Cells normally divide at the crypt (basal) region of the cell -- not the apical end.
• For each dividing cell, one daughter will become an epithelial cell and migrate toward apical surface, while the other will remain a crypt cell.
• In Celiac Disease, this process does not occur normally.

M-PHASE PROMOTING FACTOR (MPF):

• Xenopus Oocyte MPF Levels:
• Oocyte: MPF level is low, in order to freeze egg in prophase, and to prevent mitosis.
• Mature Newly Laid Egg: MPF Level is high
• Early Embryo: MPF levels alternatively high in M-Phase and low in Interphase.
• STRUCTURE: It has two subunits
• CYCLIN: The regulatory subunit. It is produced at a constant rate in the cytoplasm.
• CDC2: The kinase subunit. It phosphorylates targets to induce mitosis.
• CELL DIVISION CYCLE:
• Pre-MPF is an inactive form of Cyclin + CDC2 is sitting around in cytoplasm.
• Active-MPF is made by a combination of two things:
• Kinase Cascade from signal transducers modifies the Pre-MPF in complex reactions (multiple phosphorylations) to active MPF.
• Cyclin levels accumulate in the cytoplasm, as cyclin is continually made in many cell types.
• Mitosis is induced by Active MPF, via the catalytic activity of the cdc-2 subunit.
• Active MPF also produces cyclinases -- cyclin degradation enzymes that lower the levels of cyclin.
• This inactivates MPF, until cyclin is resynthesized or until it accumulates again in the cytoplasm.

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SARCOMERE COMPONENTS:

• Z-Disk: The union of two actin heads.
• It demarcates the sarcomere.
• At the Z-Disk, there is no myosin.
• A-Band: The distance of one thick filament, consisting of two myosin filaments.
• I-Band: The distance from the end of one thick filament to the beginning of the next thick filament.
• During contraction, the I-Band becomes shorter.
• The I-Band consists entirely of actin.
• The I-Band marks the margins of two adjacent sarcomeres. Each I-Band technically lies within two sarcomeres.
• H-Zone: The distance from the end of one thin filament to the beginning of the next thin filament.
• During contraction, the H-Zone becomes shorter.
• The H-Zone consists entirely of myosin.
• The H-Zone lies completely within the sarcomere, near the center of the sarcomere.

ACTIN MYOSIN INTERACTION: In a myofibril, in cross section:

• Six actins can interact with each myosin. Actins are in a hexagonal array.
• Three Myosins can interact, in triangular fashion, with each actin.

SKELETAL MUSCLE CONTRACTION: Myosin plays the role of an ATPase Actin-Binding Motor Protein.

• We will start with myosin bound to actin. When Myosin is bound to Actin, ATP is bound to the myosin head.
• With ATP bound, Myosin can then detach from the actin thin filament.
• Once detached, the myosin is free to hydrolyze the bound ATP to ADP + P_i. It hydrolyzes the ATP, and the ADP + P_i remain attached to the myosin head.
• The myosin then reattaches to the thin filament.
• Reattachment leads to the release of the P_i group, which in turn strengthens the interaction between the actin and myosin.
• Power Stroke: With the ATP gone, the myosin head undergoes a conformational change, causing the actin and myosin to move relative to each other.
• Then the myosin head releases the ADP.
• Then Another ATP must bind to the myosin, in order for the myosin to release from the Actin to start another cross-bridge.
• If there is no more ATP, Rigor Mortis results, in which the muscle is stuck in the contractile state, with myosin bound to actin.

REGULATION OF THE CROSS-BRIDGE CYCLE: Regulation is according to intracellular levels of Calcium and is mediated by Troponin Complex and Tropomyosin.

• RELAXED STATE:
• Tropomyosin is bound to the thin filament around its major groove, in the absence of calcium.
• The Troponin Complex is periodically bound to the thin filament such that it blocks the interaction between Actin and Myosin.
• CONTRACTED STATE
• Calcium binds to the Troponin Complex, causing a conformational change in Troponin-C.
• Troponin Complex (Troponin plus tropomyosin) removes itself from the thin filament as a result, such that Myosin can bind.

ORGANIZATION OF MUSCLE:

• MUSCLE: A whole muscle is surrounded by an epimysium membrane.
• It is composed of a bundle of fasciculi.
• FASCICULUS: Each fasciculus is surrounded by a perimysium membrane.
• It is composed of a bundle of myofibers.
• MYOFIBER (MUSCLE FIBER): Each muscle fiber is surrounded by an endomysium membrane.
• It is composed of a bundle of myofibrils.
• It is a very long and thin single muscle cell.
• It has a sarcolemma plasma membrane, with an endomysium basement membrane beyond that.
• MYOFIBRIL: A bundle of myofilaments, stacked neatly next to each other such that the Z-Disc is lined up.
• Every Thin filament in a myofibril can interact with 3 thick filaments.
• Every thick filament in a myofibril can interact with 6 thin filaments.
• Each Myofibril is bathed in sarcoplasm and surrounded by a sarcoplasmic reticulum from whence it gets it calcium supply.
• MYOFILAMENT: A very long, continuous series of sarcomeres, consisting of actin and myosin.
• Thin Filament: Actin
• Thick Filament: Myosin
• Intermediate Filament: Some muscle fibrils also have some intermediate filaments.

SKELETAL MUSCLE CROSS-SECTION (Location of Nuclei): The nuclei are all pushed to the periphery, because the actin/myosin fibers take up the central part.

• Compare this to cardiac muscle, whose nuclei are in the center.

CARDIAC -VS- SMOOTH MUSCLE: Cardiac muscle has nuclei centrally located and relatively more cytoplasm than smooth muscle.

T-TUBULES: They run in the triad, with sarcoplasmic reticulum on either side, in between each of the individual myofibrils. They transmit the Ca⁺² depolarization from the plasma membrane to the SR, which in turn transmits it to all the fibers.

• Ca⁺² release from the SR initiates the muscle contraction.
• Ca⁺² is pumped back into SR to restore resting, by a Ca⁺²-ATPase.

NEUROMUSCULAR JUNCTION:

• Active Zone: Electron-dense (dark in EM scan) patch of membrane at the end of a nerve, right at the neuromuscular junction.
• Note that vesicles are found right at the membrane, while mitochondria are found more proximal, away from the active zone.
• Junctional Fold is right opposite the active zone.
• Ach Receptors on the muscle membrane are highly concentrated right at the nerve terminal.

MUSCLE DEVELOPMENT:

• Mesenchymal cells form myoblasts.
• Myoblasts proliferate and form myotubes by fusing together, resulting in a large multinucleate cell.
• So, muscle becomes multinucleated by the fusing together of primitive myoblasts.

SATELLITE CELLS: These cells lie squeezed in-between the endomysium (basement membrane) of a myofibril and the fibers themselves.

• Developmentally they have the same origin as myotubes. They are myoblasts that did not fuse with other myoblasts during development.
• FUNCTION = Muscle Repair. They proliferate to repair damaged muscle tissue.
• They will divide to regenerate muscle, but the regeneration may be incomplete.
• MUSCLE REGENERATION:
• When the muscle fibers are gone, all that is left is the basal lamina and reticular formation of the endomysium.
• The satellite cells then migrate into the empty endomysium.
• Macrophages come in to remove necrotic remnants (debris)
• Muscle regeneration may be incomplete (muscle atrophy or weakness).
• Fiber Splitting can occur, where the satellite cell can generate smaller duplicated myofibril sections from one original myofiber.

DUCHENNE MUSCULAR DYSTROPHY: Poor function and structure of skeletal muscle.

• Symptoms / Prognosis:
• Hypertrophy of lateral thigh and calf, except that it is not muscle -- it is fatty tissue.
• Death by respiratory failure, usually due to infection and or regurgitation.
• Esophagus malfunction: The skeletal muscle portion of the esophagu1s doesn't function right, leading to problems with swallowing and regurgitation.
• Upper third of esophagus: skeletal muscle
• Middle third of esophagus: Transition of half skeletal and half smooth muscle.
• Lower third of esophagus: Smooth muscle.
• Gower's Sign: Diagnostic test of ability to squat down and stand back up.
• Histopathology: You see necrotic muscle fibers, that ultimately fill with fat infiltrates, giving the pseudohypertrophic appearance to the muscle.
• Pathology: Faulty Dystrophin Gene, resulting focal lesions on the muscle membrane ------> Calcium leaks in the cell ------> perpetual contraction ------> necrosis
• You get contracted myofibers.
• You get swollen mitochondria.
• The fibers remaining (that are not necrotic) are spheroid.
• GENETICS: X-Linked recessive disorder. It is passed from Mother to Son (hemizygous) on the X-chromosome.
• DMD Gene, coding for Dystrophin, is very large. Many of the mutations are new mutations.
• There are brain and cardiac isoforms of the Dystrophin protein.
• Werdnig Hoffman Muscular Dystrophy: Variant wherein a small portion of the dystrophin is missing. In DMD, a large portion is missing.
• DYSTROPHIN: Function is to link the muscle fibers with the extracellular matrix. It function in a spectrin-like fashion, to connect the extracellular matrix with muscle actin. This provides muscle membrane stability. Beyond that function is unclear.
• TREATMENT METHODS:
• Satellite Cell Replacement
• They tried to inject donor satellites to provide donor dystrophin, but the dystrophin couldn't get past the basement membrane barrier to get to the membrane. Using collagenase for this purpose helped but didn't increase muscle strength.
• Viral Infection with the Correct Gene -- severe limitation here was the huge size of the DMD gene.
• Repair Point Mutations on mRNA -- Novel approach where they repair the mRNA to get past the stop codon point, suppling an artificial amino acid at that point.
• In-Vitro Screening: Extract cells from the embryo and test for a particular exon on the DMD gene
• If the embryo had the DMD gene, then a positive PCR product would be obtained (i.e. some of the exons were not there).
• If the embryo did not have the DMD gene, then a negative PCR product was obtained, and they could reimplant the embryo for development.

PENNIFORM MUSCLE: Muscles with a central tendon, used for strength and stability. Example = Transversus Abdominis.

FUSIFORM MUSCLE: Muscles with a tendon on either side longitudinally, used for speed. Example = Biceps Brachii.

Ways of Distinguishing CARDIAC MUSCLE -vs- Smooth Muscle:

• Cross-Section: Cardiac Muscle has a centrally placed nucleus, whereas the nucleus is around the periphery in skeletal muscle.
• Longitudinal Section: Cardiac muscle appears striated, but with branches.
• The cardiac cells are branched in longitudinal section.
• The cardiac cells have the same structural units as skeletal muscle, although SR and T-Tubules won't be as regular.
• In Cardiac Cells you get a diad instead of a triad -- one SR membrane will adhere with one T-Tubule.

INTERCALATED DISK: The junctional complex that separates cardiac muscle cells. They always coincide with the Z-Line of muscle fibers.

• Fascia Adherens is the basic structural connections between the two cells.
• They are similar to desmosomes but are only found in cardiac cells.
• The Fascia Adherens apparently binds thin filaments in adjacent I-bands to the plasma membrane of cardiac cells.
• Desmosomes: The tightest point of connection between two cardiac cells.
• Gap Junctions: Allows fast electrical conduction between two cardiac cells.

CARDIAC ISCHEMIA:

• Structural changes in ischemia:
• 15 minutes: Structure changes occur.
• 30-60 minutes: The cell can still recover.
• > 60 minutes: The cell dies, necrosis.
• Reperfusion Injury: Occurs when oxygen is suddenly replenished after extended deprivation. It can cause mitochondria to swell up and explode.
• Histopathology of Cardiac Ischemia:
• Chromatin is more condensed than normal.
• Mitochondria swell
• Glycogen stores are absent.
• Unlike skeletal muscle, cardiac muscle cannot regenerate.

SMOOTH MUSCLE:

• Histological Characteristics
• Single central nucleus, but the amount of cytoplasm is less as compared to cardiac muscle, i.e. the nucleus takes up a great space in the cell in smooth muscle.
• Cell is not striated, as actin and myosin are not arranged in linear fashion.
• The amount of actin is greater than that of myosin. Actin is bound to dense bodies in the cytoplasm, which are held in place by intermediate filaments.
• CONTRACTION:
• RELAXED STATE:
• Myosin thick filaments are sparse, i.e. they are not polymerized.
• Myosin is dephosphorylated when relaxed.
• CONTRACTED STATE:
• Myosin Light Chain is phosphorylated.
• Myosin forms more thick filaments
• This allows the dense bodies to move toward each other.
• PROCESS OF CONTRACTION / REGULATION
• Calcium activates Calmodulin Complex.
• Calmodulin Complex then activates the Myosin Light Chain Kinase (MLCK).
• Myosin Light Chain Kinase then phosphorylates the myosin light chains
• DOWN-REGULATION: Here are ways of inducing relaxation or lessening contractile tonicity.
• beta-Adrenergic transduction can phosphorylate the Light Chain Kinase, thus deactivating it ------> No Phosphorylation of Myosin Light chains ------> Less contraction.
• Phosphatases remove the phosphate from the myosin light chain to induce relaxation.

ACTIN-BASED MOTILITY:

• Pseudopod Movement: Cytoplasmic streaming as mediated by actin polymerization and depolymerization. No myosin is involved.
• Cytokinesis: Once again involves interaction of actin and myosin to pinch the cell.

MICROTUBULE BASED MOTILITY: Dynein and Kinesin

• Dynein is a minus-end protein. It travels from plus to minus and thus aids in retrograde axonal transport.
• Kinesin is a plus-end protein. It travels from minus to plus and thus aids in anterograde axonal transport.

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COMPONENTS OF CONNECTIVE TISSUE:

• Fibers
• Collagens
• Elastic Fibers
• Ground Substance (Proteoglycans)
• Cells
• Macrophages
• Mast Cells
• Fibroblasts

COLLAGEN: The primary fiber found in connective tissue. Although other elastic fibers are also found.

• Tropocollagen is the basic structural unit, consisting of three alpha-chains arranged in a helix.
• Tropocollagen shows a typical banding pattern on EM, due to the staggered helices. Procollagen doesn't show the banding pattern.
• Chemistry:
• Every third residue is glycine.
• Hydroxyproline and Hydroxylysine are also prevalent.
• Synthesis:
• Registration Peptide: The registration peptide, distinct from the signal peptide, accomplishes two things.
• It keeps the collagen helix soluble in the cell.
• It allows the alpha-strands to align properly in the cell, in order to form the helix.
• alpha-strands are synthesized in the ER as usual. The signal peptide is cleaved but the registration peptide, as above, remains.
• Post-Translation Modifications:
• Lysyl Hydroxylase and Prolyl Hydroxylase hydroxylate lysine and proline residues.
• Various glycosylations are done.
• Procollagen is formed intracellularly. It is the soluble, spontaneously formed helix that results from the individual strands, after post-translation modifications are made:
• Procollagen still has the registration peptides intact.
• Procollagen is secreted.
• Procollagen Peptidases then cleave the registration peptide extracellularly, to result in Tropocollagen.
• Tropocollagen then forms fibrils spontaneously, stabilized by cross-links.
• Lysyl Oxidase turns on Hydro lysine residues into aldehydes, to stabilize cross-link formation.
• Fibers form by the association of fibrils.
• Collagen Types:
• Collagen I: Skin + Bone
• Collagen II: Cartilage
• Collagen III: Aorta (Reticular Fibers)
• These are also associated with elastic fibers
• A silver stain will only stain reticular fibers, so they can be identified.
• Collagen IV: Basement Membrane
• Basement membranes retain the registration peptide.
• As a result they don't form fibers but instead form sheets.
• COLLAGENASE: Breakdown of Collagen
• Process of Collagen Degradation:
• Collagenase is secreted as a proenzyme and is activated by other proteases.
• It cleaves at a specific site -- about 25% of the way down the molecule.
• The specific cleavage results in the spontaneous denaturation of the collagen helix. The smaller pieces have a lower melting point and are more volatile.
• Other proteases then finish off the job.
• Collagenase activity is temperature and fluid-dependant
• REGULATION of COLLAGENASE:
• Tissue Inhibitors of Metalloproteases (TIMPs): They bind only to activated collagenases, thus moderating their activity through negative feedback.
• Extracellular Proteases: Three types of extracellular proteases aid in the degradation of collagen:
• Metalloproteases. Collagenase is a metalloprotease
• Serine Proteases. For example -- elastase and thrombin
• Cathepsins.
• Collagen-related disorders
• Ehlers-Danlos Syndromes: Hyperextensibility of skin and joints.
• Osteogenesis Imperfecta
• Recessive Dystrophic Epidermolysis Bullosa: Too much collagenase.
• Scurvy: Vitamin-C deficiency leads to malfunctioning prolyl hydroxylase.

ELASTIC FIBERS:

• Arrangements of elastic fibers: They can be arranged in three different ways
• Fibers / Fiber Bundles -- as in skin
• Lamellae (sheets) -- as in vasculature
• Fine Networks -- as in the lung
• Protein Composition:
• Microfibrillar Protein: Forms the underlying "scaffolding" over which the elastin is laid.
• Elastin: The amorphous, elastic material.
• Elastin is resistant to degradation, except by elastase.
• Desmosine and Isodesmosine: Cross-link elastin, forming a network, and stabilizing the elastin during stretching and compressing.
• Synthesis:
• First, microfibrillar protein lays down the scaffolding.
• Then, elastins get laid down on top.
• AGING: Wrinkles occur as microfibrillar structure is lost
• Emphysema: Loss of elasticity in lung. Rare form = congenital malfunction of elastase in lung.

GROUND SUBSTANCE: Proteoglycans. They consists of a core protein + Glucosaminoglycans

• Glycosaminoglycans (GAGs): Linear polymers of repeating disaccharides of hexosamine plus a uronic acid such as glucuronic acid.
• GAG-residues are often sulfated.
• SIGNALING FUNCTION:
• GAGs have a high negative charge and are highly hydrophilic.
• Basic Fibroblast Growth Factor (BFGF) can bind to proteoglycans to promote the growth of fibroblasts.
• In this capacity proteoglycans also act as a sieve controlling passage of materials through the ECM. This property is especially important in the kidney.
• Aggrecan: Found in Hyaline Cartilage.
• Perlecan: Found in Basement Membrane
• Syndecan: Found in Epithelial Tissue. It remains attached to the plasma membrane.
• Hyaluronic Acid: Not associated with a core protein itself, but other proteoglycans can associate with it.
• Tissue Distribution:
• Vitreous humor of eye.
• Synovial Fluid of joints.
• It facilitates cell migration during growth and repair.
• Hyaluronidase is secreted when hyaluronic acid is no longer needed.

BASEMENT MEMBRANES: Made of the Basal Lamina + Reticular Lamina, or two layers of basal lamina. It is visible at the light microscope level, while basal lamina by itself is not.

• Basal Lamina: It provides a substrate for epithelial cells. It consists of different components:
• Lamina Rara: Primary constituent of the basal lamina, composed of two proteins -- laminin and fibronectin. It is directly adjacent to the epithelial cells.
• It is electron lucent in the electron-microscope.
• Laminin: Very large protein it three chains. There are specific binding domains for collagen and heparin.
• Entactin is often associated with Laminin.
• CANCER: Laminin will hook to integrin receptors. In addition it may have its own receptor, which acts in tumor metastasis.
• Fibronectin: Two chains. It is important for wound healing and cell migration.
• There are three forms of fibronectin:
• Plasma Fibronectin: Binds fibrin and fibrinogen, and plays a role in blood clots.
• Cell Surface Fibronectin
• Matrix Fibronectin -- insoluble matrix fibrils.
• Again, it has specific binding domains for heparin and collagen, and it will hook into cellular integrin receptors.
• Lamina Densa: The next layer, underneath the Lamina Rara. Composed mainly of Collagen IV (basement membrane collagen) and Heparin.
• It is electron-dense in the EM microscope.
• Again, Collagen IV still has its globular registration peptide, so it forms meshworks instead of fibers.
• Heparin Sulfate interacts electrostatically with the Collagen IV.
• Lamina Reticularis: The next layer down. Composed of Collagen III and Collagen VII. This makes up the Reticular (elastic) fibers in some basement membranes.
• Collagen III is the main reticular collagen.
• Collagen VII acts as an anchor, to hold the reticular fibers to the basal lamina.
• FNXN: The reticular lamina connects the basal lamina to the underlying stroma.
• Basement Membrane: The very bottom layer of the epithelial layer.
• Integrins: Epithelial Cellular receptors that allow the cells to interact with the basement membrane.
• STRUCTURE: Integral membrane heterodimeric proteins, with alpha and beta subunits non-covalently linked.
• Ligand-binding Domain: Binds to a specific sequence on laminin and fibronectin in the extracellular matrix.
• The specific sequence is Arg-Gly-Asp (RGD)
• Intracellular Attachment: The protein is attached to the actin cytoskeleton, via the following anchor proteins:
• Talin
• Vinculin
• alpha-Actinin
• FUNCTION: Integrins mediate cellular adhesion and migration through the ECM.

LEUKOCYTE MIGRATION: Part of the inflammatory response.

• Selectins: Specialized glycoproteins on endothelial cells, that serve to attract leucocytes to that location when activated.
• They allow for stronger interaction of the ECM with the leucocyte integrins.
• Cell Adhesion Molecules (CAMs): After being attracted by selectins, the leucocytes interact with CAMs on the endothelial surface.
• The leucocytes binds to the endothelial cell CAMs.
• Activated leucocytes must then secrete proteases and collagenases to migrate through the vessel wall and go to the site of infection.

WOUND HEALING:

• Plasma Fibronectin binds to the blood clot, thus causing Platelet Derived Growth Factor to be released by the platelets.
• PDGF, along with C₅a, then attract neutrophils and macrophages.
• Macrophages then secrete proteolytic enzymes for fibroblasts and smooth muscle cells, so they can get through the debris.
• Then the matrix is restored by fibroblasts, then the endothelial cells are restored.

TUMOR METASTASIS: Some tumor cells secrete collagenase, thus breaking down basement membranes and allowing the metastatic cells to penetrate the blood vessels.

FIBROBLASTS: RESIDENT (always present) Connective tissue cells that synthesize collagen, elastin, and basal lamina.

• Fibroblasts are not the only cells that synthesize this stuff. Epithelial tissues and smooth muscle cells can make their own ECM, too!
• Histology:
• They have little cytoplasm and lots of ER and Golgi, which is what we'd expect for their synthetic roles.
• Fibroblast Activating Factor up regulates ECM production in fibroblasts.
• Lymphocytes and monocytes can secrete fibroblast activating factor toward this end.

ADIPOCYTES: A RESIDENT CELL in connective tissue -- i.e. it is always present.

• White Adipose Tissue: Efficient, low-density storage form for energy.
• It is highly vascularized and innervated.
• HISTOLOGY: Big lipid droplet with nucleus plus minimal cytoplasmic components all off to one side.
• Lipid Deposition (Anabolic): Lipoprotein Lipase frees two of the three fats from triacylglycerols from chylomicrons in the blood.
• The lipoprotein lipase is located in the vascular endothelium.
• The remaining monoacylglycerol stays in the blood and goes back to liver.
• The two freed fatty acids diffuse through the capillary endothelium ------> basal lamina ------> connective tissue ------> adipose basal lamina ------> adipocyte ------> and into the adipose tissue.
• Lipid Mobilization (Catabolic): Hormone Sensitive Lipase is activated via the beta-adrenergic pathway. It frees fatty acids from triacylglycerols in the adipose tissue.
• beta-Adrenergic Pathway means that Hormone Sensitive Lipase is phosphorylated to be activated (via cAMP ------> Protein Kinase, etc.)
• Brown Adipose Tissue: Specialized for thermoregulation.
• It is present in hibernating and newborn humans, but not in human adults.
• Uncoupling Protein uncouples the oxidation of Acetyl-CoA in adipocyte mitochondria, such that no ATP is produced. Instead, the generated electrochemical gradient is dissipated as heat.

OBESITY:

• Hyperplasia of adipocytes occurs after birth, but the adult doesn't gain or lose adipocytes appreciably. Obesity occurs by hypertrophy of adipocytes.
• Body Mass Index = (Weight (kg)) / (Height (m))²
• Healthy BMI is 23-28.
• Two forms of obesity:
• Android Obesity, weight in upper body and abdomen, is correlated with risks for CHD.
• Gynecoid Obesity, weight in hips and thighs, is not correlated with risks for CHD.
• "Reduced Obese": When an individual gains weight and then loses it again, several things change physiologically which make it difficult to keep off the weight:
• Metabolic needs go down from the original baseline level -- i.e. total daily caloric requirements go down after having lost weight.
• Upregulation of adrenoreceptors occurs -- making it easier to mobilize fatty acids from adipose tissue (that is actually good news).
• BUT, there is a decreased response to hypoglycemia -- catecholamines aren't released as readily.
• LEPTIN: A protein made by adipocytes that correlates with obesity in laboratory mice
• EXPTs in mice suggested that obesity might be due to a lack of leptin. Mice that were obese had no leptin.
• Unfortunately this did not hold the same for humans. Obese humans actually had more leptin, so there was a positive correlation.
• There appears to be Leptin receptors in the hypothalamus, which will be involved with hunger regulation.
• They have also found leptin receptors in the choroid plexus of ventricles.
• ADIPSIN: It forms Acyl-Stimulating Protein (ASP) which generally promotes the building of triacylglycerols.
• Many obese patients have elevated adipsin levels, meaning that they can make fats readily but they have normal or subnormal rates of mobilizing them.
• Tumor Necrosis Factor: Obese patients also seem to have elevated levels of this factor. This is related to development of insulin resistance.

MAST CELLS: TRANSIENT Connective Tissue Cell. They function in allergic reactions.

• They respond to IgE from plasma cells.
• Histology:
• They characteristically have cytoplasm full of dark-staining granules.
• Mast Cell Granules are released in an allergic reaction. They contain:
• Heparin, an anticoagulant.
• Histamine, vasodilates small vessels, causing increased microperfusion of the tissue (i.e. redness)
• Serotonin
• Leukotrienes

MACROPHAGES: TRANSIENT Connective Tissue Cell. They are derived from monocytes circulating in the blood.

• Phagocytosis is often mediated by IgG
• Histology:
• Can be distinguished from other transient cells because they usually have foreign materials ingested.
• They have numerous small lipid droplets (vacuoles)

PLASMA CELLS: TRANSIENT Connective Tissue Cell. They secrete antibodies.

• Morphology / Histology:
• They have a clock-face nucleus.
• They have a perinuclear clear area.

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