BIOL 1020: Unit 4 Pt. 1: Cytoskeleton [FINAL]

cytoskeleton network main components

1. 3 well-defined filamentous structures that form an elaborate interact + dynamic network

   1. actin filaments (AKA microfilaments), microtubules, intermediate filaments 

actin filaments

1. basic building block of microfilaments; actin proteins polymerize to form actin FILAMENTS

2. solid, thin structures often organized into a branching network, many of which associated w/ the plasma membrane  

3. most abundant protein in most cells 

4. major contractile protein of muscle cells; major protein in majority of eukaryotic cells

actin in the presence of ATP

polymerizes to form a flexible, helical filament

actin structure

• The actin globular subunits (G-actin) in a filament (F-actin) are all oriented in the same direction; they have a (−)/pointed end and a (+)/barbed end. Therefore, actin is polar.

• barbed/plus end grows faster than pointed/minus end

  • growth diffs due to diffs in critical concentration

critical concentrations

the specific concentrations of actin monomers required for polymerization and growth of actin filaments.

1. require different minimal concentrations of ATP-actin monomers to elongate -> measure of this = CRITICAL CONCENTRATION (CC) 

2. CC of barbed/plus end is much lower than the pointed/minus end = barbed/plus end continues to elongate at lower ATP-actin concentrations than the pointed end can 

actin polymerization 

1. nucleation (lag phase), in which initial actin subunits come together is energetically unfavorable

   1. bc dissociation rate of actin subunits is higher than the association rate = high chance of falling apart until you have enough actin subunits 

2. once initial actin subunits come together (NUCLEATOR), actin filaments grow at faster rate after bypassing initial nucleation/lag phase

ATP hydrolysis of ATP-actin complex in vitro (how does actin age?)

1. over time, actin hydrolyzes ATP -> ADP + releases inorganic phosphate = actin aging 

2. more ADP on pointed (minus) end that’s slower growing 

   1. differentiates them thru concentration of ADP on either end  

   2. critical concentration favors ADP-actin at minus-end, rather than pointed-end 

how does actin undergo BRANCHED nucleation?

1. arp2-arp3 complex (branched actin nucleator) 

   1. function 

      1. binds laterally to “mother filament” (original actin filament that’s alr there) 

      2. acts as nucleator for new actin filaments to form relative to mother filament, which can serve as template for another one using arp ⅔ complex 

      3.  

         

      4. aged actin filaments broken, recycled to create new actin filaments 

   2. pointed (minus) end of actin binds to arp ⅔, allowing the barbed (plus) to grow faster outwards to extend the actin branch

how does actin undergo NON-BRANCHED/STRAIGHT actin nucleation?

1. only polymerize STRAIGHT actin filaments (rather than branches) 

2. how

   1. binds to actin monomers (2) -> creates formin dimer that holds 2 actin monomers tgt = forms nucleus -> formin mediates two actin subnits binding -> actin filament forms 

   2. formin moves upwards along plus end as actin subunits aggregate upwards, thus elongating the actin filament 

      

actin function

• support cell structure and position organelles properly internally

• mediate cell mvts (i.e., cilia, flagella) to propel the cell by exerting force

actin’s role in cellular migration

1. involves coordinating several proteins to extent the front of the cell (leading edge) 

   1. leading edge at front of cell is actin-rich 

   2. requires dynamic actin capable of rapid assembly, disassembly, and re-assembly facilitated by actin-binding proteins 

myosins

1. directional motors that move along actin filaments 

   1. have DIRECTION; most walked towards barbed (plus) end 

   2. function

      1. actin filament polarity (plus, minus ends) -> recognizes diff orientation of actin subunit in filament -> associates to actin filament directionally 

      2. “walks along” + moves actin filaments by hydrolyzing ATP as ther E src 

myosin II

form of myosin responsible for muscle contraction

myosin II structure

• dimeric protein with two heavy chains and two pairs of light chains, forming a distinct structure with a coiled-coil tail and two globular heads (N-terminus) that interact with actin filaments

  • other end is C-terminus

• each myosin horizontally antiparallel to each other in clusters = creates myosin II bipolar thick filament in muscle 

myosin II function in muscle contraction

1. myosin II sides along actin filaments to drive muscle contraction (in myofibrils of muscle fiber cells) 

2. actin filaments tethered to contractile proteins -> as myosin motors walk along actin -> pulls on contractile protein domain 

3. 

4. space between Z-disks = 1 contractile unit 

   1. motor heads (containining actin-binding domain that binds to actin) walk toward pointed ends of actin on bond sides -> pulls on Z disk structures on both sides -> contracts muscle

   2. motor hydrolyzes ATP -> conformational change -> power stroke where the motor moves to another actin subunit down the actin filament 

      1. motor bound to actin is bound v TIGHTLY after one round of contraction  

         1. ATP needed to dissociate so that contraction cycle can happen again (i.e., rigor/hardened muscle after death)

2. elastic proteins extend when pulled + attached to Z disk 

What is the structural organization of a skeletal muscle fiber, from cell-level features to sarcomere banding?

• Cell-level structure

  • Skeletal muscle cells are unusually large, cylindrical, and multinucleated

  • ~10–100 µm thick and >100 mm long

  • Formed by embryonic fusion of many mononucleate myoblasts

  • Called muscle fibers due to size and multinucleation

  • Among the most orderly structured cells in the body

• Fiber → myofibril organization

  • Each muscle fiber is a cable composed of hundreds of parallel myofibrils

  • Myofibrils run longitudinally through the cell

• Myofibril → sarcomere organization

  • Myofibrils consist of repeating sarcomeres (contractile units)

  • Each sarcomere extends from Z line to Z line

  • Repeating sarcomeres create the striated (banded) appearance

• Sarcomere banding pattern (EM-visible)

  • Caused by partial overlap of thin (actin) and thick (myosin) filaments

  • I band: light; contains only thin filaments

  • A band: dark; corresponds to full length of thick filaments

  • H zone: lighter region at center of A band; contains only thick filaments

  • Overlap regions (within A band): contain both thin and thick filaments

sliding filament model of muscle contraction

Core Mechanism: Muscles contract by shortening sarcomeres. Myosin heads (cross-bridges) bind to actin and undergo a power stroke, pulling actin filaments toward the sarcomere center. The filaments themselves do not change length; they slide past one another.

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Visual Changes (The "Banding" Rules):

• A Band: Remains constant (matches length of thick filaments).

• I Band & H Zone: Both decrease in width and can disappear.

• Z Lines: Move inward toward the A band.

microtubule (MT) structure

1. rigid = stronger than actin 

2. formed from tubulin heterodimers (alpha + beta) that arrange in head -> tail manner that form PROTOFILAMENT 

   1. has plus end + minus end 

      1. plus end grows faster 

      2. determined by how quickly they hydrolyze GTP -> allows for conformational change -> able to bind to new tubulin heterodimers 

   2. 12-13 protofilaments arrange in cylindrical structure = microtubule

   3. 

3. GTPases 

MT heterodimer structure

1. Free tubulin exists as an αβ-dimer, with the α-subunit binding a trapped and nonhydrolyzable GTP and the β-subunit binding an exchangeable and hydrolyzable GTP. Most of the β-tubulin in microtubules is bound to GDP. In growing microtubules, the (+) ends are capped by GTP-β-tubulin.

2. plus end = fast growing (row of GTP-beta-tubulin); minus end = slow growing

3. structural polarity of microtubules is important factor in their growth + function in directed mechanical activities for the cell 

MT dynamic instability

1. changing between rapid growth + shrinking (catastrophe); happens intrinsically to microtubules (beneficial for cell processes, like division) 

2. growth 

   1. happens faster at plus end due to higher rate of GTP hydrolysis 

      1. creates GTP-tubulin dimer/GTP-capped end 

   2. GTP cap prevents the less stable region of microtubule section containing GDP-tubulin dimers from dissociating/shrinking 

   3. during growth periods, tubulin dimers are added faster than their GTP is able to be hydrolyzed → cap of GTP-dimers on MTs at protofilament plus ends, which favors addition of more MT heterodimer subunits

3. catastrophe 

   1. random loss of GTP cap -> rapid shrinkage -> gain of GTP-tubulin dimer cap -> rapid growth 

   2. GTP hydrolysis changes subunit conformation + weakens binding affinity in the polymer -> protofilament starts curving -> depolymerization 

      1. exchanging GDP w/ GTP “straightens” out the protofilament

purpose of MT dynamic instability

1. provides mechanism where plus ends of MTs can rapidly explore the cytoplasm for appropriate sites of attachment

2. allows cells to respond rapidly to changing cell conditions that req remodeling of microtubular cytoskeleton (ex. MT disassembly + remodeling into bipolar mitotic spindle) 

how are MTs organized

1. centrosome = main MT-organizing centers (MTOCs) where MTs typically NUCLEATE in animal cells

   1. minus end attached to nucleating sites (gamma-tubulin ring complexes) on the surface of centrosome; plus ends grow outwards (forming a radial array)

      1. radial array usually in undfferentiated cells that contain centrosome near nucleus 

      2. cell differentiation -> dissolution/disassociation of centrosome into “bare components” that nucleate MTs -> non-radial form 

   2. surrounded by pericentriolar material + has 2 centrioles inside

   3. 

basal bodies + other MTOCs 

1. centrosomes not the only MTOCs 

2. outer MTs in cilium or flagellum generated direct from MTs in structure called basal bode (resides @ base of cilium or flagellum) 

all MTOCs share common protein component =

gamma-tubulin

how do MTs branch?

augmin (protein complex) recruits branching MT to nucleate off of existing MT thru gamma-tubulin ring complex

how can MT organization change according to the cell?

1. diffs along cell types after differentiation (which lends MT organization to the cell’s function)

2. undifferentiated cells usually have a radial array of MT where they come out from centrosomes, whereas differentiated cells have much more organized MT networks

types of MT-associated proteins

• AKA MAPs

  • +TIPs

  • structural MAP proteins

    • microtubule associated proteins

+TIPs

• proteins that recognize + associates w/ growing plus ends of MT to connect them w/ other structures (i.e., membranes) 

  • moves along w/ MT as they grow 

  • localizes at plus end “tip” of MTs to recognize the end of MT; continuously associates to MT as long as it grows 

• doesn't associate w/ MTs that aren’t growing 

• can track +TIP proteins using GFP to see only the MT plus end tips that are actually growing

structural MAP proteins

• crosslink MTs; by linking 1 to another MT 

• can also associate w/ itself + act as the structure that brings 2 MTs tgt

• MAP4 and MAP2

MAP4

1. crosslinks + creates gap/spacing between MTs most optimally for cell function

2. more MAPs = more crosslinking/connection between MTs

3. 

   1. in absence of MAP4, MT gets bunched tgt 

4. zippering of anti-parallel microtubules is important for MT organization in skeletal muscle cells

MAP2/Tau family of structural MAPs

length of projection domains of MAPs between MTs in specific cell times determines the spacing of the MTs in the cell + thus cell structure/cytoskeleton

MT motors

• kinesins

• dynein

kinesin

• motor protein that walks along MTs

• motor usually walks PLUS END-DIRECTED (N-termius), very few minus end-directed 

• motor in the middle doesn’t walk; depolarizes MT + controls MT growth 

  1. MT depolymerase, where it favors GDP-binding/catastrophe

kinesin 1 structure

1. dimer 

2. 2 head motor domains w/ coiled-coin tail domain connected to light chains

kinesin 1 function

1. moves along MT thru ATP hydrolysis; ATP hydrolyis undocks kinesin, allowing it to take another step 

2. most processive motors = walks along MT w/o diassociating for long period of time; bc at least 1 motor domain is always held on the MT (as opposed to only having 1 motor domain that undocks often) 

3. often holds vesicle as it moves

dynein 

1. super fast

2. is a monomer, so can’t innately walk along MT by itself processively 

   1. most associate w/ another dynein monomer to move 

   2. minus end-directed MT motor 

cytoplasmic dynein

encoded by a single gene in almost all eukaryotes but absent in flowering plants + some algae