Classes- Cell Tissue Biology Flashcards
How many cells do we have?
Body = 3.7210^13
Brain = 1.710^11
What is the size of a normal cell? Diâmetro and volume mass?
Sphere volume (4/3)pir^3; cell density: 1.1g/ cm^3
Cell and tissue microscopic observations:
in vivo, ex vivo, Staining, Fixation, Sectioning
Fixation and Sectioning of samples for microscopy
Tissues should be cut (using a microtome) in slices thin enough to avoid complete absorbance of the incident ligth or electrons.
Fixation aims to denature and cross-links groups on adjacent molecules of proteins and nucleic acids, rendering them insoluble and stable for subsequent procedures and observation.
Staining
Modifying a substrate into colored product or into a precipitate by an endogenous enzyme
Immunocytochemistry/ immunohistochemistry
Dyes (colorimetric, electron-dense or fluorescent) have a low and nonspecific affinity for biological molecules, but they can bue chemically coupled to antibodies specific for almost any desired protein (and other macromolecules).
FRET
FRET is a mechanism describing energy transfer between two chromophores
Optogenetics
Involves the use of ligth to control neurons that have been genetically modified to express ligth- sensitive ion channels.
Cytoskeleton
Provides structural support and shape for the cell, organizes cytoplasm (polarity) and permits directed movement of organelles, chromossomes, and the cell itself. Also, througth association with extracellular matrix and others cells it stabilizes tissues
Extracellular matrix (functions and composition)
Functions: mechanical strength, adhesion, migration, chemical selectivity, proliferation, differentiation, apoptosis and cell shape. All of them depend on ECM composition and density.
Composition:
Structural proteins: e.g. collagens and non-collagenous proteins (e.g. elastic)
Specialized proteins: e.g. fibronectin and laminin
Glycosaminoglycans ( GAGs): polysaccharides
Proteoglycans: a protein core attached to GAGs
Tight junctions
Ribbon-like bands connecting adjacent cells that prevent leakage of fluid across the cell layer
Are formed by interactions between strands of transmembrane proteins (occludin and claudins) on adjacent cells.
Gap junctions
consist of assemblies of six (x2) connexins (>20 types), which form open channels through the plasma membranes of adjacent cells where some ions and small molecules pass through.
Stable cell-cell junctions mediated by the cadherins. Interactions between cadherins mediate two types of stable cell- cell adhesions:
In adherens junctions, the cadherins are linked to bundles of actin filaments via catenins.
In desmosomes, desmoplakin links members of the cadherin superfamily ( desmogleins and desmocollins) to intermediate filaments.
Both mechanical and biochemical molecules influence ECM dynamics in multiple ways
By realeasing small bioactive signaling molecules, releasing growth factors stored within the ECM , eliciting Structural changes to matrix proteins which expose cryptic sites and by degrading matrix proteins directly.
Signal transduction
The overall process of converting signals into cellular responses, as well as the individual steps in this process.
Lipophilic signaling molecules
Retinoic acid acts through Hox genes, which ultimately control embryonic anterior/posterior patterning in early developmental stages.
Thyroxine is involved in controlling the rate of metabolic processes in the body and influencing physical development.
Cortisol is involved in response to stress and anxiety. It increases blood pressure and blood sugar, and reduces immune responses.
Progesterone is involved in the female menstrual cycle, pregnancy and embryogenesis of humans and other species.
Estradiol has not only a critical impact on reproductive and sexual functioning in females, but also affects others organs including the bones.
Testosterone is the principal male sex hormone and an anabolic steroid.
Hydrophilic signaling molecules
Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, and stopping use of fat as an energy source.
Glucagon is released when blood glucose levels start to fall too low, causing the liver to convert stored glycogen into glucose and release it into the bloodstream.
Growth factors are diverse group of molecules capable of stimulating cellular growth.
Epinephrine is released into the bloodstream in response to physical or mental stress, as from fear or injury . It initiates many bodily responses, including the simulation of heart action and an increase in blood pressure, metabolic rate, and blood glucose concentration.
Histamine triggers the inflammatory response by increasing the permeability of the capillaries to white blood cells and proteins.
Serotonin
Acetylcholine
Erythropoietin
Interferons
G-proteins-coupled receptors (GPCRs)
Are a class of cell-surface receptors that activate G-proteins
Intracellular second messages
Are intracellular signaling molecules that greatly amplify the original first messenger signal
May be coupled downstream to kinase/phosphatase cascades
Protein kinases and phosphatases
Are involved directly or indirectly in the signal transduction from cell surface receptors
Can be regulated by second messengers
Microfilaments
The actin cytoskeleton is organized in bundles and networks of filaments which are held together by actin cross-linking (or branching).
Actin also interacts with motor proteins (myosins).
Actin contributes for the shape (biconcave disk) of the erythrocyte by interacting with other proteins (cytoskeletal and integral proteins)
Microtubules
Intracellular membrane vesicles travel along microtubules
Kinesin is (+) end- directed microtube motor protein (anterograde transport)
Dynein is (-) end-directed microtubule motor protein (retrograde transport)
Collagens
-Types I, II and III are the most abundant and form fibrils of similar structure.
-Type IV forms a two-dimensional reticulum and is a major component of the basal lamina.
-Collagens are mainly synthesized by fibroblasts but epithelial cells also synthesize them.
-“Collagens” is the major protein comprising the ECM (and also from the animal kingdom).
Laminin and fibronectin
Laminin and fibronectin form bridges between structural ECM molecules, and connect the ECM to cells and to soluble molecules within the extracellular space.
Fibronectins (dimers) bind many cells (via RGD-integrins) to fibrous collagens and other ECM molecules.
Laminin and type IV collagen form the 2-D network of basal lamina.
Glycosaminoglycans (GAGs)
Hyaluronic acid, dermatan sulfate, chodroitin sulfate, heparin, heparatan sulfate, and keratan sulfate;
-are long unbranched polysaccharides, highly negatively charged, containing a repeating disaccharide unit: N-acetylgalactosamine (GaINAc) or N-acetylglucosamine (GIcNAc), and a uronic acid such as glucuronate or iduronate.
GAGs confer high viscosity to the solution and low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrety to cells and provides passageways between cells, allowing for cell migration.
Hyaluronic acid is unique among the GAGs in that it does not contain any sulfate and is not found covalently attached to proteins as a proteoglycan. It has very large molecular weight and can displace a large volume of water.
Proteoglycans
Are proteins linked to GAGs (also called mucopolysaccharides).
- GAGs extend perpendiculary from the core in a brush-like structure. The linkage of GAGs to the protein core involves a specific trisaccharide which is linked to the protein core through an O-glycosidic bond to a S or T residue in the protein.
G-proteins (trimeric and monomeric GTPase switch proteins)
In their on state bind and regulate the activity of effector proteins.
Operational model for ligand-induced activation of effector proteins associated with GPCRs
1-Binding of hormone induces a conformational change in receptor
2-Activated receptor binds to Galfa subunit
3-Binding induces conformational change in Galfa; bound GDP dissociates and is replaced by GTP; Galfa dissociates from G(beta-gama)
4-Hormone dissociates from receptor; Galfa binds to effector, activating it
5-Hydrolysis of GTP to GDP causes Galfa to dissociate from effector and reassociate with G(beta-gama)
GPCRs that regulate adenylyl cyclase
In the liver, glucagon and epinephrine bind to different receptors, but both receptors interact with and
activate the same Gsa, which activates adenylyl cyclase, thereby triggering the same metabolic responses.
Positive and negative regulation of adenylyl cyclase activity occurs in some cell types, providing fine-tuned control of the cAMP level. For example, stimulation of adipose cells by epinephrine, glucagon, or ACTH activates adenylyl cyclase, whereas prostaglandin* PGE1 or adenosine inhibits the enzyme.
PKA
cAMP-activated protein kinase A (PKA) mediates various responses in different cells - alters the transcription of specific genes or the activity of specific proteins- by phosphorylation of serine and threonine residues in the target proteins (X-R-(R/K)-X-(S/T)-F).
At low concentrations of cAMP, the PKA is an inactive tetramer. Binding of cAMP to the regulatory (R) subunits causes a conformational change in these subunits that permits release of the active, monomeric catalytic (C) subunits. cAMP binding is cooperative.
GPCRs that activate gene transcription by a kinase cascade
Kinase cascade that transmits signals downstream from mating factor receptors in S. cerevisiae.
The receptors for yeast a and a mating factors are coupled to the same trimeric G protein. Ligand binding leads to activation and dissociation of the G protein. In the yeast mating pathway, the dissociated Gbg activates a protein kinase cascade analogous to the cascade downstream of Ras that leads to activation of MAP kinase. The final component, Fus3, is functionally equivalent to MAP kinase (MAPK) in higher eukaryotes. Association of several kinases with the Ste5 scaffold contributes to specificity of the signaling pathway by preventing phosphorylation of other substrates.
GPCRs that activate phospholipase C (PLC) via Ca 2+ and PKC
Synthesis of DAG and IP3 (diffusible) from membrane-bound phosphstidylinositol (PI).
The level of PIs are regulated by extracellular signals, especially those that bind to receptor tyrosine kinases or cytokine receptors.
IP3/DAG pathway and the elevation of cytosolic Ca 2+
This pathway can be triggered by ligand binding to certain G protein-coupled receptors and several other receptor types, leading to activation of phospholipase C. Cleavage of PIP2 by phospholipase C. Cleavage of PIP2 by phospholipase C yields IP3 interacts with opens Ca 2+ channels in the membrane of the endoplasmic reticulum, causing release of stored Ca 2+ ions into the cytosol. One of various cellular responses induced by a rise in cytosolic Ca2+ is recruitment of protein kinase C (PKC) to the plasma membrane, where it is activated by DAG. The activated Kinase can phosphorylate various cellular enzymes and receptors, thereby altering their activity. As endoplasmic reticulum Ca 2+ stores are depleted, the IP3-gated Ca2+ channels bind to and open store-operated TRP Ca2+ channels in the plasma membrane, allowing influx of extracellular Ca 2+.
GPCRs that activate phospholipase C (PLC) via Ca2+ and calmodulin
The Ca2+/calmodulin complex regulates the activity of many different proteins (e.g. cAMP-phosphodiesterase, nitric oxide synthase) and kinases (e.g. GPK that hydrolyses glycogen) and phosphatases that control the activity of various proteins including transcription factors.
Regulation of contractility of arterial smooth muscle by nitric oxide and cGMP
Nitric oxide (NO) is synthesized in endothelial cells in response to acetylcholine and the subsequent elevation in cytosolic Ca2+. NO diffuses to nearby smooth muscle cells where activates an intracellular NO receptor with guanylyl cyclase activity. The resulting rise in cGMP leads to activation of protein kinase G (PKG) (which indirectly induces dephosphorylation of myosin), relaxation of the muscle, and thus vasodilation. (Nitroglycerin (is converted to NO by mitochondrial aldehyde dehydrogenase) and “Viagra” inhibit a cGMP-phosphodiesterase)
GPCRs that activate a PDE (phosphodiesterase)
The high level of cGMP present in the dark acts to keep cGMP-gated cation channels open; the light-induced decrease in cGMP leads to channel closing, membrane hyperpolarization (cell interior more negative), and reduced neurotransmitter release
Operational model for rhodopsin-induced closing of cation channels in rod cells
In dark-adapted rod cells , a high level of cGMP keeps nucleotide-gated nonselective cation channels open. Light absorption generates activated opsin, O* , which binds inactive GDP-bound Gt protein and mediates replacement of GDP with GTP. The free Gt(alfa) GTP generated then activates cGMP phosphodiesterase (PDE) by binding to its inhibitory gama subunits and dissociating them from the catalytic alfa and beta subunits. Relieved of the inhibition, the alfa and beta subunits convert cGMP to GMP. The resulting decrease in cytosolic cGMP leads to dissociation of cGMP from the nucleotide-gated channels in the plasma membrane and closina of the channels. The membrane then becomes transiently hyperpolarized.
GPCRs that regulate ion channels
Operational model of muscarinic acetylcholine receptor in the heart muscle plasma membrane.
The exit of K+ results in hyperpolarization of the cell membrane that slows the rate of muscle contraction.
Mechanisms of GPCR signal termination (3 pathways)
- hydrolysis of GTP to GDP, which is accelerated by GAPs and RGSs, causes Gα to dissociate from effector and bind with Gßɣ
- upon phosphorylation of the GPCR by protein kinases (PKs) or GPCR kinases (GRKs) arrestin binding may occur preventing G-protein coupling as well triggering the process of receptor internalization through clathrin-mediated endocytosis
Intracellular receptors
Small lipophilic molecules like steroids (cortisol, progesterone, estradiol, testosterone), thyroxine and retinoic acid diffuse across plasma membrane and interact with intracellular receptors altering gene expression at transcription or post-transcription level. Long term stimulus (hours to days).
Sequential activation of gated ion channels at a neuromuscular junction
Arrival of an action potential at the terminus of a presynaptic motor neuron induces opening of voltage- gated Ca2+ channels (step 1) and subsequent release of acetylcholine, which triggers opening of the ligand-gated
nicotinic receptors that are ion channels in the muscle plasma membrane (step 2). The resulting influx of Na+ produces a localized depolarization of the membrane, leading to opening of voltage-gated Na+ channels and generation of an action potential (step 3). When the spreading depolarization reaches T tubules, it triggers opening of voltage-gated Ca2+-release channels and release of Ca2+ from the sarcoplasmic reticulum into
the cytosol (step 4). The rise in cytosolic Ca2+ causes muscle contraction
TGFb Receptors and the Direct Activation of Smads
■ Stimulation by TGFb leads to activation of the intrinsic serine/threonine kinase activity in the cytosolic domain of the type I (RI) receptor, which then phosphorylates an R-Smad, exposing a nuclear-localization signal.
■ After phosphorylated R-Smad binds a co-Smad, the resulting complex translocates into the nucleus, where it interacts with various transcription factors to induce expression of target genes.
■ TGFb signaling generally inhibits cell proliferation. Loss of various components of the signaling pathway contributes to abnormal cell proliferation and malignancy.
TGFß* receptors
Receptor Serine Kinases That Activate Smads
- have serine/threonine kinase activity
- directly activate Smads and then the transcription of PAI-1
TGFb-Smad signaling pathway.
Step 1a: In some cells, TGFb binds to the type III TGFb receptor (RIII), which presents it to the type II receptor (RII). Step 1b: In other cells, TGFb binds directly to RII, a constitutively phosphorylated and active kinase. Step 2: Ligand-bound RII recruits and phosphorylates the juxtamembrane segment of the type I receptor (RI), which does not directly bind TGFb. This releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI between the membrane and kinase domain. Step 3: Activated RI then phosphorylates Smad3 (shown here) or another R-Smad, causing a conformational change that unmasks its nuclear- localization signal (NLS). Step 4: Two phosphorylated molecules of Smad3 interact with a co- Smad (Smad4), which is not phosphorylated, and with importin b (Imp-b), forming a large cytosolic complex. Steps 5 and 6: After the entire complex translocates into the nucleus, RanGTP causes dissociation of Imp-b. Step 7: A nuclear transcription factor (e.g., TFE3) then associates with the Smad3/Smad4 complex, forming an activation complex that cooperatively binds in a precise geometry to regulatory sequences of a target gene. Shown at the bottom is the activation complex for the gene encoding plasminogen activator inhibitor (PAI-1).
Transforming growth factor beta (TGF-β)
is a protein that controls proliferation, cellular differentiation, and other functions in most cells
Receptor Tyrosine Kinases and Activation of Ras
Receptor tyrosine kinases (RTKs), which bind to peptide and protein hormones, may exist as preformed dimers or dimerize during binding to ligands.
Ligand binding leads to activation of the intrinsic protein tyrosine kinase activity of the receptor and phosphorylation of tyrosine residues in its cytosolic domain. The activated receptor also can phosphorylate other protein substrates.
Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like G protein, Ras cycles between an inactive GDP-bound form and an active GTP-bound form.
Ras cycling requires the assistance of two proteins, a guanine nucleotide–exchange factor (GEF) and a GTPase-activating protein (GAP).
Cytokine receptors and receptor tyrosine kinases
- share many signaling features
- the phosphotyrosines of the receptors are docking sites for proteins (with SH2 domains)
General structure and ligand -induced activation of receptor tyrosine kinases (RTKs) and cytokine receptors
The cytosolic domain of RTKs contains a protein tyrosine kinase catalytic site, whereas the cytosolic domain of cytokine receptors associates with a separate JAK kinase (step 1). In both types of receptor, ligand binding causes a conformational change that promotes formation of a functional dimeric receptor, bringing together two intrinsic or associated kinases, which then phosphorylate each other on a tyrosine residue in the activation lip (step 2). Phosphorylation causes the lip to move out of the kinase catalytic site, thus allowing ATP or a protein substrate to bind. The activated kinase then phosphorylates other tyrosine residues in the receptor’s cytosolic domain (step 3). The resulting phosphotyrosines function as docking sites for various signal- transduction proteins
Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs).
The receptors for epidermal growth factor (EGF) and many other growth factors are RTKs. The cytosolic adapter protein GRB2 binds to a specific phosphotyrosine on an activated, ligand-bound receptor and to the cytosolic Sos protein, bringing it near its substrate, the inactive RasGDP. The guanine nucleotide–exchange factor (GEF) activity of Sos then promotes formation of active RasGTP. Note that Ras is tethered to the membrane by a hydrophobic farnesyl anchor
Kinase cascade that transmits signals downstream from activated Ras protein to
MAP kinase.
In unstimulated cells, most Ras is in the inactive
form with bound GDP; binding of a ligand to its RTK or cytokine receptor leads to formation of the active RasGTP complex (step 1).
Activated Ras triggers the downstream kinase cascade depicted in steps 2–6, culminating in activation of MAP kinase (MAPK). In unstimulated cells, binding of the 14-3-3 protein to Raf stabilizes it in an inactive conformation. Interaction of the Raf N-terminal regulatory domain with RasGTP relieves this inhibition, results in dephosphorylation of one of the serines that bind Raf to 14-3-3, and leads to activation of Raf kinase activity (steps 2 and 3). Note that in contrast to many other protein kinases, activation of Raf does not depend on phosphorylation of the activation lip. After inactive RasGDP dissociates from Raf, it presumably can be reactivated by signals from activated receptors, thereby recruiting additional Raf molecules to the membrane.
