Cell Bio Exam 3

nuclear envelope

• double membrane/bilayer structure, inner and outer nuclear membranes with a perinuclear space

• an extension of the ER

• supported by a framework of proteins

nuclear pores

• cluster together/localized on nuclear envelope

• very large, ~120 by 85 nm and weight ~120-130 mD

• composed of many proteins, well over 1,000

• octagonal symmetry, fiber fish basket structure

• <= 40 kD in size and <= 10 nm molecules freely diffuse through pores

  • small proteins and ions

  • microinjected gold particles of this size = size exclusion barrier

• selective active transport for larger particles

  • larger gold particles coated with proteins found in nucleus can enter because they have a NLS, not cleaved off

nucleoplasm

the contents of the nucleus, contains chromatin

nuclear import requirements

• need 3 things:

  1) NLS

  2) importin

  • beta subunit grabs onto a protein with a NLS

  • alpha subunit interacts with the NPC

  • a.k.a a nuclear transport receptor

  3) Ran

  • small monomeric GTPase

• in vitro reconstitution- what are the minimal requirements for moving a protein into the nucleus?

  • fluorescently labeled BSA conjugated to a NLS would move into the nucleus

  • mixed Ran and importin and BSA (cargo) together and could reconstruct the event but could not with just Ran and BSA or BSA and importin

nuclear import mechanism

• importin bound to cargo with NLS enters nucleus, interacts with Ran which facilitates release of cargo from importin (conformational change), Ran bound to importin exported from cell, Ran disassociates from importin via GAP

• concentration gradient- high Ran-importin complex concentration in nucleus but low in cytoplasm

  • maintained by Ran GEF in nucleus → spatial restriction

nucleoporins

• 500 to 1000 nuclear proteins, combinations of 30 types of polypeptides

• structural and functional

• polypeptide domains rich in FG repeats form intrinsically disordered domains in NPC → mesh of interactions creates a hydrogel barrier for larger objects

  • importin has surface AAs that preferentially interact with hydrogel AAs, breaking noncovalent linkages forming hydrogel so it can bring proteins in

nucleolus

• essentially a biomolecular condensate of the nucleus

• defined region with defined molecules, excludes other molecules, localized functional events

• vary in size and number depending on species, cell type, and state of cell

• fibrils- where you find DNA that encodes for rRNA

• dense fibril- rRNA being encoded plus imported RNPs

nuclear export mechanisms

• exportin (nuclear transport receptor) binds with cargo that has a NES and exits nucleus, interacts with Ran which facilitates release of cargo from exportin (conformational change), Ran disassociates from exportin via GAP

• mRNAs coated with export proteins can move through the NPC

• viruses can change the shape of the nuclear envelope to exit the nucleus and enter the cytoplasm

nuclear lamina

• made up of proteins called lamins

• form a meshwork of filaments just beneath the inner nuclear membrane

• provide primary structural support to the nucleus

  • experiment with lamin mutant protein formed irregularly shaped nucleus

nucleoplasmic reticulum

• extensions of the nuclear envelope

• irregularities in surface topology

• nuclear envelope (often the inner membrane) can extend into cytoplasm or nucleoplasm

• nuclear envelope (both inner and outer membrane) can invaginate to form pockets of cytoplasm in nucleoplasm

functions of the nuclear envelope

1) compartmentalization- barrier between nucleoplasm and cytoplasm, nucleus has distinct molecular population

2) site of transport- NPCs

3) platform for signaling

4) structural integrity- nuclear lamina

5) regulation of gene expression

organization of the nucleoplasm/chromatin

• DNA usually associated with proteins in nucleus = chromatin

• histones- octameric proteins that DNA wraps around, forming a nucleosome (transcriptionally inactive)

• nucleosomes associate to form chromatin fibers

• highest order of packing structure called a chromatin compartment or territory

  • each contains the DNA of a single chromosome

  • Boveri noticed nuclei of roundworm cells have dense protrusions, non-homogenous objects within the nucleus → chromatin territories

  • thought experiment in 70s- if you track DNA damage less chromosomes damaged if chromatin is compartmentalized, actual experiment found damaged chromosomes in specific territories

fluorescence in situ hybridization (FISH)

use small fluorescently labeled nucleic acid sequence with homology/base pair complementarity to DNA in chromosome they are interested in studying → chromosome painting

heterochromatin vs. euchromatin

• three ways to define these terms:

  1) functional definition- heterochromatin = transcriptionally inactive, euchromatin = transcriptionally active

  2) cytological definition- dark staining, dense outer regions = heterochromatin, light staining, less dense inner regions = euchromatin

  3) structural definition- level of compaction (how much DNA brought together irrespective of its level of organization/packing)

• transcription occurs in specific regions of the nucleus called transcriptional hubs or neighborhoods

• position of a gene change when transcribed

  • positional effect- change position of gene, gene is in a different transcriptional state

  • active in euchromatic positions, inactive in heterochromatic positions

  • occurs via diffusion

how does organization arise in the nucleus?

• drivers

  • protein-protein interactions drive polymerization of chromatin

  • homotypic clustering- like molecules like to associate with like molecules

• constraints

  • nuclear lamina has proteins associated with it that interact with proteins associated with the chromatin

  • nuclear bodies- biomolecular condensates, hold on to strands of DNA and keep them in a fixed position, dozens of types in a nucleus

    • nucleolus- site of rRNA production and ribosomal subunit assembly)

    • Cajal bodies- assembly of complexes required for mRNA processing

    • speckles- storage of complexes required for mRNA processing

• rule exception- DNA close to nuclear pores will be transcribed even though in outer part of cell

signal transduction

the conversion of signals from one form to another

eicosanoids

phospholipid derivative, important in the inflammation response specifically platelet aggregation

4 classes of signaling

1) paracrine signaling- short distance signaling, presynaptic cell releases molecule that binds to postsynaptic cell

2) autocrine signaling- signaling molecules bind receptors on same cell

3) endocrine signaling- long-distance signaling, hormones move through bloodstream

4) juxtacrine signaling- molecules on the surface of one cell bind to receptors on the surface of another cell, physical contact, closest form of cell-cell signaling

receptor locations

• signal receptors are on the plasma membrane or are intracellular

  • many hormones bind intracellular receptors to regulate transcription

flow of information during signal transduction utilizing cell surface receptors

• ligand binds to receptor → receptor undergoes a change (in state, phosphorylation) → activation of biochemical pathways via signal transduction which utilizes signaling molecules and second messengers that can affect cellular processes and gene expression

• signals and pathways can be integrated (one signal activates two pathways, two signals activate same pathway, crosstalk)

response speed

• signals altering protein function are fast

• signals altering gene expression are slow

3 classes of cell surface receptors

1) ion-channel-linked receptors- ligand binds to channel and allows ion channel to open

2) G-protein-linked receptors

3) enzyme-linked receptors

heterotrimeric G-proteins and GPCRs

• interact with a GPCR

• activated when ligand binds GPCR

• GPCR spans membrane 7 times, intracellular domain serves as binding site

• alpha, beta-gamma subunits

• upon ligand binding GPCR intracellular domain changes conformation so inactive G-protein binds, receptor stimulates exchange of GDP for GTP on alpha subunit, alpha subunit separates from beta-gamma and signals for downstream events

• to shut off, intrinsic hydrolyzation of GTP

• active alpha subunit binds to transmembrane enzyme, which catalyzes the conversion of a substrate to a second messenger (cAMP, cGMP, IP3, DAG)

second messenger pathways

• active alpha G-protein subunit activates adenylyl cyclase, which converts ATP into cAMP

  • phosphodiesterase converts cAMP into AMP to eliminate the secondar messenger

  • cAMP binds to protein kinase A (PKA) that phosphorylates proteins in the cytoplasm and nucleus, can turn on transcription

• active alpha G-protein subunit activates phospholipase C, which cleaves PIP2 into two secondary messengers, IP3 and DAG

  • DAG remains associated with the plasma membrane, activates protein kinase C (PKC), which phosphorylates substrates

  • IP3 binds to ER calcium ion channels, which release calcium ions into the cytoplasm, calcium ions bind to PKC and other calcium-dependent proteins to regulate them

enzyme-linked receptors

• ligands dimerize a receptor, a ligand dimerizes receptors, ligands dimerize receptors

• receptor tyrosine kinases closely associates and autophosphorylates when dimer ligand binds

• adaptor protein SH2 domain bind phosphorylated tyrosine in RTK and SH3 domain binds to proteins enriched in prolines (GEF)

• GEF activates small monomeric G-protein Ras, which initiates a mitogen-activated protein kinase cascade (MAP)

  • scaffold protein holds three kinases together to allow for rapid signaling

regulation/adaptation of cell signaling

1) specificity of receptor

• for ligand

• for effector (target)

2) abundance of ligand or effector (turnover)

• ensure response is appropriate duration and amplitude

3) modulation by inhibitors

antagonists and agonists

• synthetic molecules that mimic naturally occurring ligands

• antagonist- blocks receptor from binding to ligand, inhibits a response

• agonists- mimics ligand and induces a response

ligand-receptor binding

• requires molecular complementarity

• amount of time ligand spends bound to receptor depends on the ratio between the forward and reverse reactions (kreverse/kforward)

• dissociation constant = [R][L]/[RL] or kreverse/kforward

  • low Kd = high affinity between receptor and ligand

  • high Kd = low affinity between receptor and ligand

• maximum physiological response may not require binding of all receptors on cell surface

• fewer receptors require a high ligand concentration to elicit the same cellular response

number of receptors on cell surface?

• labeled ligand (insulin) with I125, measured amount bound to cell surface

• redid experiment using cells not expressing insulin receptor, found that a little insulin could bind to cell surface in absence of insulin receptor → nonspecific binding

• total binding - nonspecific binding = specific binding (number of receptors), ~34,000 receptors per cell

• receptor Kd > free ligand concentration to allow a dynamic range of responses to varying ligand concentrations

signal desensitization mechanisms

• receptor sequestration- endocytose receptor into cell, reversible via exocytosis

• receptor down-regulation- endocytose and degrade receptor

• receptor inactivation- block intracellular receptor signaling

• signaling protein inactivation- downstream inhibitory molecule

• inhibitory protein production- autoinhibition

signaling pathways- toxins and ordering

• cholera toxin effect on GPCR pathways

  • prevents Gsalpha GTP from hydrolyzing → always on → massive amount of cAMP produced → loss of water from body (dehydration)

• pertussis toxin

  • locks Gialpha in GDP state → off → massive amount of cAMP produced → loss of water from body (dehydration)

• double mutant analysis determines order of steps in a signaling pathway

  • non-functioning mutant X plus constitutively active Ras → signaling does occur → X before Ras

  • by combining a loss of function mutation with a gain of function mutation, able to establish pathway order

  • non-functioning mutant Y plus constitutively active Ras → signaling does not occur → Y after Ras

mechanisms to transmit signals between cells

1) diffusible, extracellular signals (autocrine, paracrine, endocrine)

2) cell surface-bound, extracellular signal (juxtacrine)

3) releasable membrane-associated signals (exosomes, microvesicles)

4) diffusible, intracellular signals (junctional synapses - gap junctions, nanotubes, plasmodesmata)

releasable membrane-associated signals (exosomes and microvesicles)

• exosomes

  • small vesicles

  • released in large numbers

  • unique lipid composition in membrane

  • specific protein or mRNA contents- packets of information

  • bind to or fuse with target cell membrane

  • formed by invagination of multivesicular endosome (MVE)

• microvesicles

  • large vesicles

  • released in small numbers (unless cancer cell)

  • formed by pinching off from source cell membrane

physical properties of the cytoplasm

1) finite volume (1 × 10^-11 uL to 0.5 mL, depends on cell size)

2) concentrated (200-300 mg protein/mL)

3) complex, partially defined solution (~20,000 different proteins)

4) non-homogenous solution

5) active matter (molecular and thermal motion)

6) characteristics of both gel and solution

7) viscoelastic (elastic = quick return of state after displacement, viscous = slow return of state after displacement, viscoelastic = quick displacement but slow return of state)

8) low microviscosity/ high macroviscosity (free diffusion only if < 10 nm diameter)

evidence for a cytomatrix

1) particle diffusion rates (indirect immunofluorescence and GFP revealed cytoskeleton)

2) microscopy (non-ionic detergent + EM = cytoskeleton artifact)

cytoskeleton three filament networks

1) intermediate filaments

• vimentin, ~10 nm

2) microtubules

• tubulin, 25 nm

3) microfilaments

• also found in nucleus

• actin, 7 nm

microtubules

• hollow cylinders of variable length

• assembled from tubulin subunits

• hallmark of eukaryotic cells

• involved in many cellular functions

main functions of microtubules

• chromosome segregation in mitosis and meiosis

• transport of membrane-bounded organelles

• cell motility and morphogenesis

• ciliary and flagellar beating

structure of microtubules

• subunit tubulin, composed of alpha and beta proteins tightly bound to one another → heterodimer

• alpha binds GTP for structural support, beta covers alpha GTP and binds to GTP itself, which can be hydrolyzed

• if [tubulin] > 10 mM, bind to each other (alpha binds to beta) → protofilaments

• lateral protofilament interactions → microtubule

• singlet, doublet (cilia, flagella), triplet (basal bodies, centrioles)

critical concentration (Cc) of assembly for tubulin

• critical concentration = 10 mM

  • above = tubulin polymer, below = tubulin dimers

  • plateau → total mass of microtubules remains constant

• microtubules have intrinsic polarity + and - end indicating a difference between the ends

  • + end where more growth occurs when [tubulin] > Cc

    • used pre-existing microtubules to seed microtubule assembly in vitro

    • Cc lower for + end than - end

  • polarized arrays- + ends near membrane/end of cell, - ends near center of cell

microtubule dynamic instability

• microtubule shrinking = catastrophe, microtubule growth = rescue

  • at + end, on rate GTP tubulin > off rate GTP tubulin and off rate GDP tubulin > on rate GDP tubulin (GTP = on, GDP = off)

  • some of GTP-bound tubulin hydrolyzes GTP into GDP over time → subunits come off (catastrophe)

  • GDP-bound tubulin regenerate into GTP-bound tubulin → higher [GTP-bound tubulin] → subunits add on (rescue)

  • on rate GTP tubulin > rate GTP hydrolysis

microtubule-associated proteins (MAPs)

• some proteins called MAPs bind to either free tubulin subunits or microtubules and affect their assembly/disassembly

  • some increase microtubule stability and prevent their depolymerization

  • others like catastrophin bind to microtubule lattice and destabilize it by either promoting GTP hydrolysis or weakening lateral contacts between protofilaments

(+) TIPS

• family of proteins that are a type of MAP

• ex: EB1 kymograph

  • shows same region of interest repeated over time, overlap panels

  • EB1 always associated with microtubule when elongating → EB1 stimulating microtubule growth? wrong! no other independent experiments such as biochemical ones

  • actually promotes GTP cap hydrolysis and microtubule shrinkage

  • located past the GTP cap, not actually at the tip

do prokaryotes have cytoskeletons?

• have cytoskeletal polymers, just not tubulin and microtubules

• FtsZ dimer has incredible structural homology with tubulin, forms filament rings within a bacterium when at high enough concentration involved in binary fission

• Asgard group of archaea have microtubules made of protofilament bundles

functional classes of MAPS

1) capping proteins (nucleators/stabilizers)

• minus-end capping proteins (gamma-TURC)

• plus-end capping proteins

2) depolymerizers (destabilizers)

• (+) TIPS

• catastrophe factors (some kinesins)

• severing proteins (katanin cuts microtubules)

• heterodimer sequestering proteins (bind to GTP-bound tubulin)

3) cross-linkers (connectors/stabilizers)

• bind to microtubules to stabilize them

• to other microtubules

• to membrane of organelles or plasma membrane

4) molecular motors (movers)

• utilize free energy released by ATP hydrolysis to change conformation of protein and allow its movement

• plus-end directed motors (kinesin protein family)

• minus-end directed motors (dynein protein family)

kinesin structure

• dimer, two heavy chains twist to form a tail plus light chain

• two polypeptide globular heads, where ATP hydrolysis and microtubule binding occurs

  • ATP-bound head tightly associated and causes neck linker of other head to move in front like taking a step, loses ATP and disassociates, other head gains ATP…

processivity

• a measurement/description of how far a molecule can travel before it detaches from its substrates

  • kinesins can keep their heads on a microtubule for a distance of up to 1 micrometer

discovery of kinesin in squid axoplasm

• squids have very large axons with lots of organelles moving in both directions

• dynein (minus-end directed protein) drives movement from axon to cell body, but what drives movement from cell body to axon (plus-end directed protein)?

• started experiments with squid axoplasm extruded onto a glass slide and could observe motility → biologically active material

  • tried using same protocol used to discover dynein but chemical conditions bad for kinesins

• another experiment by Vale used movement rather than ATPase activity as the guide

  • cell fractionation and ATPase assay of axoplasm

  • took fractions and put a droplet of each on glass slides, placed microtubules on top of each slide, observe amount of motion that each fraction produced → microtubule gliding assay

  • peak of ATPase activity near fraction 22 while peak of movement near fraction 30 → only need 1 active kinesin to move an entire microtubule

  • step toward single molecule analysis

motor MAPs

• kinesins moving organelles out to edge of cell, dyneins moving organelles toward center of cell → motors have to have coordinated activity to prevent a tug-of-war

• melanophore cells have vesicle organelles called melanosomes that are filled with pigment

  • dispersal or aggregation of melanosomes determined by level of cAMP → signaling events responsible for controlling movement

microtubule organizing centers (MTOCs)

• a.k.a centrosomes, each made of two centrioles, triplet microtubules

• not found in plants, protists, or fungi

• ~80 proteins in a layered cloud that surrounds the centrioles (pericentriolar material)

• likely a biomolecular condensate- distinct properties, distinct proteins, can sequester molecules and bring larger structures together

• microtubules originate from rings in pericentriolar material (gamma-TURC)

gamma-TURC

• gamma tubulin exists at minus end of microtubules

• cell fractionation and centrifugation revealed different S values for alpha and beta tubulin versus gamma tubulin

• gamma-tubulin ring complex (gamma-TURC) serves as the site of assembly for microtubules, circular platforms

mitosis

• how cells divide/chromosomes separate

• phases: interphase, prophase, prometaphase, metaphase, anaphase, telophase/cytokinesis

• interphase: cell growth/maintenance, centrosome duplication

• prophase: centrosomes begin to separate, nuclear envelop fragments in open mitosis but not in closed mitosis (in fungi, protists, and algae, nuclear envelope becomes permeable and NPCs lose their specificity so that microtubules can assemble inside the nucleus)

• prometaphase: mitotic spindle begins to form and attach to chromosomes

• metaphase: chromosomes aligned at the metaphase plate (center of the cell)

• anaphase: chromosomes separate

• telophase: cell reestablishes normal state

• cytokinesis: cytoplasm divides

prophase

• chromatin condenses into chromosomes

• microtubules becoming very unstable → increase in dynamic instability

• centrosome separate to opposite poles of the cell

  • tetrameric kinesins on microtubules “walk” toward positive ends facing membrane, causing the microtubules to slide apart- a “pushing” force

  • “pulling” force = dynein connects plasma membrane to microtubules and pulls them toward plasma membrane

prometaphase

• nuclear envelope breakdown

• kinetochore

  • massive assembly of proteins

  • centromere region of DNA with repetitive DNA sequences that act as a site for kinetochore to bind

  • kinetochore captures microtubules by tightly binding with them when they interact, not a random event, some form of communication between the chromosome and microtubule that biases the microtubule to impact the kinetochore

• three types of microtubules

  • kinetochore microtubules

  • overlap/polar microtubules come in contact with microtubules from other side of cell

  • astral microtubules radiate outward toward plasma membrane

second way to generate a mitotic spindle without centrosomes

Ran GEF stimulates production of Ran-GTP, which facilitates the assembly of mitotic spindle by binding to importin so that it releases microtubule stabilizing factors, microtubules will arrange into a mitotic spindle with the help of motor proteins

microtubule-kinetochore interactions

• chromosomal passenger complex (CPC) located near kinetochores of sister chromatids, made up of many types of proteins like Aurora B kinase which phosphorylates kinetochore proteins

• Ndc80 proteins link microtubule to kinetochore complex proteins connected to centromeric chromatin

• when Aurora B kinase phosphorylates Ndc80, there is a weak attachment of the microtubule to the chromosome

• to get a strong attachment, remove the phosphate group added by Aurora B kinase using the enzyme protein phosphatase 1 (PP1)

• weak attachment becomes strong because tension from microtubule pulling on kinetochore complex causes Nbc80s to move away from Aurora B and toward PP1 (physical rearrangements lead to stabilization)

sister chromatid pair movement to metaphase plate

• dynein attached to kinetochore complex walking toward minus end of microtubule pulls sister chromatid pair, microtubule depolymerizing/shrinking in front to make room

• kinesin walking towards + end of microtubule pushes sister chromatid pair, microtubule growing/repolymerizing behind

• chromokinesins associated with ends of chromosomes

  pushing them toward + end of microtubule/cell center

metaphase

• motor protein forces balance out and sister chromatids hold position at metaphase plate

• microtubules maintain constant length due to treadmilling (steady state, common in microfilaments)

anaphase/telophase

• anaphase A

  • sister chromatids separating, kinetochore microtubules depolymerized by specific kinesins, at minus end depolymerization also occurring

  • dynein walks chromatid toward minus end of microtubule

  • molecular events break protein linkages between sister chromatids, come apart as kinetochore microtubules shrink

  • centrosomes same distance apart → spindle maintaining constant size overall (overlap and aster microtubules growing, kinetochore microtubules shrinking)

• anaphase B

  • spindle becomes larger, centrosomes move apart from each other due to tetrameric kinesins and microtubules becoming longer as they slide past each other

• telophase

  • reassembly of nuclear envelope

“purse-string” model of cytokinesis

• myosin II pulls F-actin ring tight

• symmetric cell division = equal, asymmetric cell division = unequal

microfilaments

• polymers of actin

• intrinsic polarity

  • used trypsin to cut the myosin motor protein, cleaving off the tail and left with two heads that retain ability to bind to microfilament

  • mix heads with microfilament, place on EM grid, coat in heavy metals → myosin heads bind and look like arrows

  • barbed end = plus end, pointed end = minus end

actin

• binds ATP, ATPase

• monomers = globular actin (G-actin)

• polymer = filamentous actin (F-actin)

• as [G-actin] increases, reach Cc where you see assembly of G-actin into F-actin

  • as the amount of actin increases, more microfilaments will form

microfilament assembly analogous to microtubule assembly

• ATP/ADP instead of GTP/GDP

• nucleation, elongation (rate of assembly greater than rate of loss), steady state/equilibrium (rate of assembly = rate of loss, net mass does not change)

• adding a nucleator (preformed polymer) to a concentrated solution of actin causes assembly to occur faster by skipping lag phase (kinetics) but does not affect Cc (thermodynamics, stabilizers lower Cc)

treadmilling- microfilament remains ~ constant length

• Ccs of both + and - end are close to each other

• researchers determined rate constants for addition or subtraction of subunit to both microfilament ends

• rate constant expresses proportionality between [molecule] and reaction rate

• dissociation constant Kd = 1/K (equilibrium constant)

  • approximates Cc (micromolar)

  • ATP-bound actin will have higher affinity for barbed end because its Kd is lower → assembly happens on barbed end before pointed end

  • free G-actin > 0.12 & < 0.6 means actin adding to barbed end but coming off of pointed end → maintaining constant length even though subunits are adding and coming off → treadmilling

actin associated proteins

• regulate microfilament dynamics, organization, and movement

• types

  • stabilizing- cross-linking, bundling and end-blocking/capping proteins

  • destabilizing- severing and depolymerizing proteins

  • sequestering proteins

  • linking- membrane-binding proteins

myosin superfamily

• actin binding protein

• 24 families

• barbed end (+) directed motor protein, except for class VI

• some are processive (remain attached to microfilament for long distances)

cross-bridge cycle

• describes how myosin “walks” along an actin filament

  • strong attachment when nucleotide-free

  • ATP binds, myosin releases

  • ATP hydrolyzed, pulled back in conformational change

  • Pi leaves, strong attachment

  • ADP leaves, power stroke back to original conformation causes movement of microfilament

• important for muscle contraction

myosin II self-assembly

• double-headed myosin

• tend to fold up on each other unless a myosin light chain kinase (MLCK) phosphorylates light chains at base of heads, resulting in myosin II assuming an elongated structure

• tail domain can interact with tail domains of other myosin IIs in an anti-parallel fashion → bipolar thick filaments

• myosin thick filament can slide anti-parallel actin thin filaments past each other

  • decrease diameter of F-actin contractile ring in cytokinesis

  • involved in muscle contraction- thin filaments bound to a Z-protein disc at their barbed ends, in middle in thin filament circle is a thick filament that walks toward barbed ends, causing sarcomeres to contract

  • sarcomere arrays form microfibrils, which are packed into a muscle cell, muscle cell bundles = a muscle

  • tropomyosin blocks myosin binding site on actin, when calcium binds one of the troponin protein complex subunits, causes a conformational change that drags tropomyosin away from the binding site so that myosin can bind the actin microfilament

  • calcium released from sarcoplasmic reticulum, which senses voltage changes in the plasma membrane of the muscle cell, depolarization causes calcium release from SR

intermediate filaments

• basic unit a dimer → tetramer → protofilament → intermediate filament

• 8 protofilaments per filament

• filaments within the same cell can be homopolymers or heteropolymers

• no filament polarity

• phosphorylation inhibits filament assembly (promotes filament disassembly)

• filaments dynamic

• no nucleotide triphosphate binding

• many different types found in multicellular organisms

  • keratins, neurofilaments, lamins

• provide mechanical strength

  • little force needed to break microtubules

  • microfilaments stiff

  • intermediate filaments can bend and maintain structural integrity under more force

  • epidermolysis bullosa simplex keratin mutation

cell-matrix interactions

• migrating cells connect via focal adhesions/contacts

  • fibroblasts

  • focal adhesions connect bundles of actin filaments with matrix

  • microfilaments → actin binding proteins → integrin (transmembrane protein) → fibronectin (ECM protein)

• stationary cells connect via hemidesmosomes

  • epithelia

  • intermediate filaments connect hemidesmosome to cell

  • intermediate filaments → plaque → integrin → laminin

extracellular matrix (ECM) of animal cells

• the “space” between cells

• constitutes a significant volume of tissues

• filled with proteins and polysaccharides

• determines physical properties of a tissue (hardness of bone, transparency of cornea)

• regulates cell behavior (cell proliferation, cell shape, cell migration)

• provides positional information

• lattice upon which cells move

• reservoir of signaling molecules

• contains three classes of molecules

  • structural proteins: elastins and collagens

    • collagen fibers large/long, provide structural support

    • elastins cross-link so that they remain connected when stretched, elasticity

  • proteoglycans (mucoproteins) form a matrix

    • large molecular sponge, negatively charged, attract water molecules and cations, resists compression in tissues

    • glycosaminoglycans (GAGs)- repeating chain of disaccharide subunits connected by a protein to form a proteoglycan

  • adhesive glycoproteins: fibronectin and laminin

    • fibronectin in migratory cells, laminin in stationary cells

    • different functional domains bind cell-surface receptors and collagen

basal lamina

• type of ECM found between epithelial cells and underlying layers of connective tissue

• about 50 nm in thickness

• basal lamina + sublayer of gelatinous matrix = basement membrane

functional classifications of cell junctions

• occluding junctions- sealing

  • tight junctions (vertebrates)

  • septate junctions (invertebrates)

• anchoring junctions/adhering junctions- stability

  • actin filament attachment sites

    • cell-cell junctions (adherens junctions)- adhere

    • cell-matrix junctions (focal adhesions)- anchor

  • intermediate filament attachment sites

    • cell-cell junctions (desmosomes)- adhere

    • cell-matrix junctions (hemidesmosomes)- anchor

• communicating junctions- channel forming

  • gap junctions

  • nanotube junctions

  • plasmodesmata (plants only)

junctional complex of intestinal epithelial cells

• tight junction (seal)

  • “molecular fence”

  • occludin and claudin major transmembrane protein components

• adherens junction (stability)

  • mechanically couple cells by tethering adjacent adhesion belts (bands of actin filaments) together

  • invagination of epithelial sheet

  • adhesion belts connected by cadherins (homophilic cadherin requires calcium ion binding)

• desmosome (stability)

  • uses cadherins to connect desmosome complexes

  • tether together adjacent bundles of intermediate filaments

cell-cell adhesion can occur in the absence of junctions

• homophilic or heterophilic interactions

• velcro principle- individually interactions are weak but are collectively strong

variables affecting adhesion

• identity of adhesion proteins

• amount of adhesion proteins

• spatial distribution

• biochemical properties (Ca2+)

• external forces (fluid flow)

gap junctions

• channels that arrange end-to-end, aligning to form a tube/pipe that spans both cell membranes, providing direct cell-cell cytoplasmic connections

• densely packed

• many found in cardiac cells

nanotubes

• provide direct cell-cell cytoplasmic connections

• discovered because HIV moves through them

amoeboid movement

• dynamic actin cytoskeleton

• gelation- cytoplasm becoming more solid

  • cross-linkers, bundling proteins, nucleators

• solation- cytoplasm becoming more liquid-like

  • capping proteins, monomer sequestering proteins, filament severing proteins, depolymerizers

cell cortex

• F-actin rich peripheral layer beneath plasma membrane

• maintains cell stability, F-actin cross-linked by actin binding proteins

• confers mechanical strength

• dynamic network regulated by actin binding protein

• cortical pressure resists hydrostatic expanding pressure

  • cortical relaxation- loss of internal actin cytoskeletal structure due to certain actin binding proteins causes cell to “ooze” forward

cell crawling

• cell is in contact with the substrate via focal adhesions

• lamellipodium- part of cell facing direction of movement

  • contains a branched network of microfilaments that extend the plasma membrane forward as they grow

    • Arp2/3 complex branches microfilaments at leading edge by acting as nucleators

      • Rho-GTP (like Cdc42) in membrane activates WASp which extends its conformation and activates Arp2/3 by binding to it

    • listeria bacteria show how actin assembly can produce movement

  • filopodium- long cytoplasmic projection from the lamellipodium that contains parallel microfilament bundles

• the rear of the cell translocates via cortical contraction and de-adhesion

  • microfilament sliding in contractile bundles pulls the cell forward

  • retrograde flow (continuous flow of microfilaments toward the center of the cell) arrested by focal adhesions

• stress fibers are contractile actomyosin bundles that create tension in the middle of the cell