Cytoskeleton: Microtubules and Filaments

Cytoskeleton Overview

• Serves as an intracellular network of protein filaments within the cytoplasm
• Consists of three fiber types: microfilaments, intermediate filaments, and microtubules
• Fibers are polymers made of small protein subunits held by noncovalent bonds

Microtubules

• Hollow, cylindrical structures approximately 25 nm in diameter found in all eukaryotic cells
• Play roles in determination of cell shape and motility
• Built from αβ-tubulin heterodimers (α-tubulin and β-tubulin), each ~50 kDa
• Microtubule-Associated Proteins (MAPs) bind to the microtubule surface, controlling its functions and regulating polymerization, dynamics, organization and cellular interactions

Microtubule Structure

• Polymers made of αβ-tubulin heterodimers in a repeated head-to-tail arrangement
• Protofilament: a linear array of tubulin heterodimers; typically 13 protofilaments form a microtubule in cells
• Microtubules consist of 11 to 15 protofilaments
• Parallel protofilaments have the same polarity, with β-tubulin subunits at one end and α-subunits at the opposite

Microtubule Polarity

• Ends designated plus or minus based on dynamic properties
• Plus ends have exposed β-subunits and grow fast
• Minus ends have exposed α-subunits and grow slow
• Singlet microtubules (13 protofilaments) are common in the cytosol
• Specialized arrays exist in doublet or triplet microtubule structures

Doublet & Triplet Microtubules

• Doublet microtubules: 10 or 11 protofilaments of a second microtubule fused to a complete 13-protofilament microtubule (cilia and flagella)
• Triplet microtubules: an extra 10 or 11 protofilaments of a third microtubule fused to the second microtubule (basal body structures)

Microtubule Organizing Center (MTOC)

• Microtubules are organized, typically near the nucleus in interphase cells
• MTOCs contain a pair of centrioles, also known as centrosomes
• Minus ends are embedded in the MTOC
• Plus ends extend away from it
• γ-tubulin (a tubulin variant) is involved in microtubule nucleation but does not incorporate into microtubules

γ-tubulin Ring Complex (γ-TuRC)

• Protein complex containing γ-tubulin and other proteins
• Nucleates microtubule polymerization
• MTOCs in higher plants lack centrioles but function as microtubule organizing centers

Microtubule Polymerization and Dynamics

• Dynamic structures constantly being polymerized and depolymerized via nucleation-elongation
• Nucleation: formation of a short microtubule nucleus facilitated by γ-TuRC capping the minus end
• Nucleation is unfavorable under normal conditions

Microtubule Elongation

• Elongation occurs rapidly after the nucleation phase
• Tubulin heterodimers add to the exposed end, causing net filament elongation
• Steady state: growth and depolymerization are balanced

Critical Concentraton

• Tubulin heterodimer concentration determines whether microtubules polymerize or depolymerize
• Above critical concentration, they polymerize, below which they depolymerize
• The number of tubulin heterodimers that add to the polymer per second depends on the concentration of the free tubulin heterodimers

GTP Hydrolysis

• Each α- and β-tubulin binds one GTP molecule
• α-tubulin bound GTP is never hydrolyzed/exchanged
• β-tubulin binds GTP, which can be exchanged and hydrolyzed
• GTP hydrolysis is accelerated when tubulin heterodimers are incorporated into filaments reducing the binding affinity of subunits

GTP Cap

• GTP hydrolysis weakens bonds between tubulin subunits, allowing depolymerization
• High concentration of tubulin heterodimers leads to rapid microtubule growth, a GTP cap encourages growth
• Microtubules with GDP-tubulin at the end depolymerize readily

Treadmilling

• The rate of subunit addition depends on the concentration of free αβ-tubulin
• Rate of polymerization/depolymerization faster at the plus end
• When αβ-tubulin concentration is higher at the plus-end but is lower at the minus-end, microtubules treadmill
• Treadmilling maintains polymer length

Dynamic Instability

• Microtubules switch between slow growth and rapid shortening phases
• Dynamic instability: result of GTP hydrolysis after assembly
• GDP-tubulin heterodimers lead to depolymerization
• GTP-bound tubulin at the tip protects it from disassembly

Catastrophe and Rescue

• Hydrolysis catches up to the tip, quick depolymerization/shrinkage occurs = catastrophe
• Adding GTP-bound tubulin to the tip again provides a cap and protects the microtubule from shrinking = rescue

Kinesins and Dyneins

• Kinesin: a motor protein that moves along microtubules, mammals encode 40+ kinesin proteins (kinesin-1 to kinesin-14)
• The position of the motor domain classifies them into N-type, M-type, and C-type kinesins
• Conventional kinesin (kinesin-1) has two heavy/light chains

Kinesin Structure and Function

• Heavy chain has a motor head, α-helical stalk, and globular tail domain
• The motor head binds microtubules and ATP
• The tail domain binds receptors on cargo membranes
• It is a plus-end directed motor involved in organelle transport

Dyneins

• Minus-end directed microtubule motors
• Composed of 2 or 3 heavy chains (with a motor domain) and light chains
• Motors exist as two-headed or three-headed structures.
• Are the largest and fastest molecular motors

Dynein and Dynactin

• Dynein cannot mediate cargo transport on its own
• Interaction with dynactin (a large complex of 11 subunits) is required which links vesicles/chromosomes to dynein light chains
• Dynein family is divided into cytoplasmic and axonemal dyneins
• Cytoplasmic dyneins mediate retrograde transport of vesicles toward the minus end of microtubules and localize the Golgi complex.

Cytoplasmic Dynein

• Contains ~12 polypeptide subunits: two identical heavy chains (homodimer) with ATPase activity and light chains
• Axonemal dyneins facilitate the sliding of microtubules (cilia/flagella)

Cilia and Flagella

• Flagella are locomotory organelles in prokaryotes and eukaryotes
• Eukaryotic flagella have a specialized arrangement of microtubules that differs from prokaryotic flagella
• Two components: a filament and a basal body (or kinetosome).
• Filament has a bundle of microtubules (axoneme): nine outer doublet microtubules surrounding a central pair of singlet microtubules (9+2 arrangement)

Cilia and Flagella Structure

• Doublet microtubule is composed of A- and B-tubules
• A-tubule contains 13 protofilaments
• B-tubule has 10 protofilaments
• Connection btwn A and B tubules has the protein tektin which is similar to intermediate-filament proteins

Connecting Structures & Dynein Arms

• Axoneme connects with the basal body
• The two central singlet and nine outer doublet microtubules run continuously
• Dynein arms (inner and outer rows) attach to the A tubule of each doublet microtubule
• Dynein interacts with the tubule of the neighboring doublet

Nexin & Radial Spokes

• Nexin links adjacent outer doublet microtubules
• Radial spokes connect from the central singlets to each A tubule of the outer doublets which is the third linkage system
• Radial spoke and central pair complexes regulate beating

Axonemal Bending

• Bending is caused by the sliding of doublet microtubules
• Arrangement: (+) end at the outer tip of the axoneme
• Dynein arms on the A-tubule slide on the neighboring doublet's B-tubule toward the (-) end
• ATP drives active sliding through successive formation and breakage of cross-bridges.

Cilia Types

• Eukaryotic flagella and cilia share common structural organization in length, number per cell, and beating pattern.
• Two cilia types: motile and non-motile
• Motile cilia beat in large numbers per cell (oviduct and respiratory tract epithelial cells, protists)
• Non-motile cilia: primary or sensory

Comparing Flagella and Cilia

• Motile cilia have an axoneme of nine doublet microtubules and a central pair of singlet microtubules (9+2 arrangement).
• Primary cilial axonemes lack the central pair of singlet microtubules
• Flagella beat with wave-like motion to drive a cell in direction
• Motile cilia produce strokes to move cells perpendicular to cilium axis

Centrioles

• Barrel-shaped structures made of microtubules found in animal cells, fungi and algae cells
• Have walls of nine triplet microtubules (9 + 0 arrangement)
• Have central proteins
• Pair of centrioles is present in MTOC (centrosome)
• Two centrioles are arranged perpendicular to each other

Centrosome

• Cloud of amorphous material surrounds a pair of centrioles
• Has a centrosome matrix (or pericentriolar material)
• Has role in bipolar spindle assembly but is unclear.
• Centriole- and centrosome-ablated cells can form spindles

Centrosome Cycle

• Duplication occurs during the interphase stage
• Centrioles and other components are duplicated
• At G1 phase, the two centrioles separate
• During S phase, a daughter centriole grows near each mother centriole at a right angle
• Completion of elongation occurs by G2
• Two centriole pairs remain close together in a complex until M phase

M Phase

• Complex splits into two
• Each centriole pair turns into a MTOC
• Array of microtubules = aster
• Two asters move to opposite sides of the nucleus
• Separated chromosomes form two poles of the mitotic spindle
• Each daughter cell receives a centrosome.

Centrosome & Chromosome Duplication Parallels

• Semi-conservative duplication is used
• Two halves separate and serve as templates for construction of a new half
• Centrosomes/chromosomes replicate once only to ensure that the cell enters mitosis with two copies

Actin Filaments

• Made of G-actin
• G (globular)-actin is a 43 kDa ATPase
• Binding site for ATP or ADP
• Highly conserved cytoskeletal protein at high concentrations in eukaryotic cells

Actin Filament Properties

• G-actin subunits join head-to-tail to make filamentous (F)-actin
• Parallel F-actins twist around each other in a right-handed helix to form a filament
• Polarity determined by regular, parallel arrangement of subunits
• Thinner, more flexible, and shorter than microtubules

Actin Filament Structure

• Polar structure with two ends: slow-growing minus and faster-growing plus
• Minus end = pointed end
• Plus end = barbed end
• Plus end polymerizes ~10 times faster than the minus end

ATP Hydrolysis

• Similar to GTP hydrolysis
• Not required to form the filament, but weakens bonds in the polymer, promoting depolymerization

G-actin Polymerization (In Vitro)

• Nucleation phase: G-actin aggregates into short, oligomers
• Elongation phase: nuclei are rapidly elongated by G-actins
• Steady-state phase: equilibrium reached between filaments/monomers
• G-actin monomers exchange at filament ends, but no net change in total length of filaments

Actin Nucleation

• Assembly requires formation of a trimeric nucleus which is kinetically unfavorable
• Proteins bypass spontaneous nucleation to nucleate actin assembly (actin nucleating proteins)
• Proteins are actin-related protein-2/3 (Arp2/3) and formins
• Arp2/3, Arp3, and five other proteins (ArpC1, ArpC2, ArpC3, ArpC4, ArpC5) make Arp2/3 complex (~220 kDa)

Arp2/3 Complex

• Found in eukaryotes
• Binds to the sides of existing filaments
• Polymerizes a new filament at a 70° angle
• Activation requires nucleation promoting factors (NPFs)
• Mammalian cells have WASp and WAVEs

Formins

• Multi-domain proteins
• Characterized by formin homology (FH) domains (FH1/FH2)
• FH1 domain facilitates filament assembly
• FH2 domain nucleates F-actin and remains associated with the barbed end
• Function is regulated by binding with Rho GTPases
• Accelerate elongation of the barbed ends of actin filaments

Drugs Affecting Microtubules & Actin Filaments

• Polymerization and depolymerization of subunits are affected

Comparison of Microfilaments and Microtubules

Actin-Targeting Drugs

• Cytochalasin D: Binds plus ends and prevents elongation
• Latrunculin: Binds G-actin monomers and prevents polymerization
• Phalloidin: Binds tightly to stabilize
• Jasplakinolide: Induces actin polymerization/stabilizes
• Swinholide: Severs filaments

Microtubule-Targeting Drugs

• Nocodazole: Causes depolymerization
• Colchicine: Binds to prevent polymerization
• Colcemid: Binds to prevent polymerization
• Taxol: Binds and stabilizes, preventing depolymerization
• Vinblastine: Binds β-tubulin to prevent polymerization
• Vincristine: Binds free tubulin to prevent polymerization

Myosin

• Actin filament-based motor protein
• Myosin-II is an oligomer of identical heavy chains and non-identical light chains
• Heavy chains possess a globular head and amino acid sequence that forms an extended coiled-coil
• Each head has an actin-binding site, a catalytic site for ATP hydrolysis

Tails

• A major role of the rodlike tail of myosin-II is to polymerize into bipolar filaments
• Long coiled-coil tails allows the molecules to polymerize orienting at the two ends of the thick filament
• When treated with the proteases trypsin, it is cleaved into light meromyosin and heavy meromyosin
• Light meromyosin is the a-helical rod

Myosin Types & Function

• Two-headed (myosin II)
• One-headed (myosin I, III, IV.... XIV)
• Unconventional myosins do not form bipolar thick filaments so they may be involved in cell movements such as the transport of membrane vesicles

Myosin Facts

• All myosins have a conserved motor domain
• Other domains vary by myosin type
• All move towards the plus end of an actin filament, besides myosin VI

Actin & Myosin Functions

• Function in eukaryotes in muscle contraction, endocytosis, cytokinesis, cell motility, cyclosis, and intracellular transport
• Crawling movements of cells across a surface represent a basic form of cell locomotion, and consists of coordinated movements
• Extension (or protrusion) of the leading edge, attachment of the leading edge to the substratum and retraction of the rear of the cell into the cell body are key

Protrusive Structures

• Different protrusive structures include filopodia, lamellipodia and pseudopodia
• The structures differ in the arrangement of actin in one, two, or three dimensions, respectively
• Protrusive structures attach to the substratum in cell locomotion
• Slow-moving cells (fibroblasts) = focal adhesions
• Rapidly moving cells (amoebas or white blood cells) = more diffuse contacts

Muscle Contraction (Retraction)

• Occurs in the retraction stage of cell crawling
• Attachments of the trailing edge to the substratum are broken
• The rear of the cell recoils into the cell body

Muscle Contraction

• Skeletal muscles attach to bone by tendons made of connective tissue as well epimysium
• Skeletal muscles are made bundles called fascicles which have perimysium
• Each fasciculus contains muscle fibers (or muscle cells) with endomysium
• Muscle fibers are striated with peripherally located nuclei.

Sarcolemma & Sarcoplasm

• Cell membrane = sarcolemma
• Sarcoplasm contains a modified ER (sarcoplasmic reticulum)
• T-tubules invaginate the sarcolemma to activate sarcoplasmic reticulum
• Sarcoplasm possesses fine thread-like contractile structures called myofibrils

Sarcomeres

• Actin/myosin make sarcomeres = functional units of a myofibril
• Actin thin filaments attach at their plus ends to the Z-disc (separation point btwn sarcomeres)
• α-actinin anchors the actin to Z-disc
• Minus ends extend toward the middle of the sarcomere, where they overlap with thick filaments

Thin & Thick Filaments

• Thin filaments contain actin, nebulin, tropomodulin, tropomyosin, and troponin
• Tropomyosin attaches to F-actin in the groove btwn its filaments
• Troponin is a complex of troponins T, I and C
• Troponin T binds to tropomyosin as well as to the other two troponin components
• Troponin I binds to actin and troponin T and inhibits the F-actin-myosin interaction
• Four molecules of calcium bind to Troponin C.

Nebulin

• Repeating 35-amino-acid actin-binding motif stretching
• Extends from the Z-disc to dictate the length of the filament
• Ends of the thin filaments are capped by tropomodulin to be stable
• Thick filaments are made by several hundred myosin molecules and associate by tail-to-tail interactions ending in 1.5 um long filaments
• Proteins hold the thick filaments together for relaxation (molecular spring)

Sarcomere Changes

• The protein titin may have a role in the relaxation of the muscle
• Sarcomere shortening: myosin filaments slide past the actin thin filaments
• Light bands of sarcomeres = I bands (isotropic)
• Dark bands of sarcomeres = A-bands (anisotropic)
• A narrow H-zone in the center has thick filaments
• M-line (supporting proteins) is at the middle

Huxley Observation

• Observation: I-band shortens during muscle contraction but thick/thin filaments do not
• Inferred thick filaments slide along the thin filaments which is described as a sliding filament model

Sarcomere Components

Electrical Action

• Signal from the nerve causes an action potential which goes through the T-tubules and sarcolemma
• T-tubules contains proteins, which are activated and opens calcium release channels
• Increase in calcium starts the contraction of each myofibril
• Myofibrils in the cell contract at the same time within millisecond

Calcium Release

• An abundant calcium ATPase pumps the calcium back into the sarcolemma rapidly
• Cytoplasmic levels are restored within 30 milliseconds, allowing the myofibrils to relax
• In resting muscle, troponin I and troponin T interact with tropomyosin to keep tropomyosin away from the groove that prevents myosin heads

Muscle Steps

1. Release of neurotransmitter at the motor end-plate
2. Action potential in muscle fibers is generated by activation of nicotinic acetylcholine receptors
3. Depolarization along T-tubules
4. Calcium gets released from the sarcoplasmic reticulum
5. Calcium binds to troponin C uncovering myosin binding sites on actin
6. Linkages between actin and myosin results in sliding of thin on thick filaments

Actin-Myosin Coupling

• Myosin head locks tightly and binding of ATP causes a conformational change to allow movement
• Hydrolysis makes a displacement along the filament
• Release of phosphate causes ATP and ADP hydrolysis, triggering a change during which the head regains its original shape
• Smooth muscle contraction: regulated by tropomyosin-troponin complex on thin actin filament that is turned on/off with calcium

Vertebrate Contraction

• Smooth muscle is regulated by light chain phosphorylation and dephosphorylation
• Muscles contract when is phosphorylated by enzymes and calcium level indirectly regulates phosphorylation
• Myosin gets activated through calcium

Smooth Muscle facts

• Smooth muscle contraction involves thick filament regulation and occurs independently from calcium.
• It gets activated by Rho kinase and light chain phosphorylation

Intermediate Filaments

• Ropelike cytoplasmic filaments (~10 nm diameter) found in metazoans (not plants/fungi)
• Proteins = chemically heterogenous & have variations in molecular weight

• 50 divided into six groups

• Structural reinforcement and organization rather than cell motility
• Individual polypeptide of intermediate filament is an elongated, non-α-helical N-terminal head domain

Protein Properties

• Made of a long, tandem repeats of a seven amino acid sequence (heptad repeat)
• Chain forms a parallel coiled coil dimeric structure
• 2 dimers form an antiparallel tetramer of four polypeptide chains
• Tetramer, the soluble subunit of intermediate filament, as well as the αβ-tubulin heterodimer or G-actin organizes (alpha-helix or beta-sheet)
• Protein subunits don't have a nucleoside triphosphate binding site

Classification of Intermediate Filament Proteins

Muscle Contraction

• Relies on actin-myosin interactions, ATP hydrolysis, and calcium regulation (cross-bridge cycle)

1. Resting State (Before Contraction)
   • Actin and myosin are not bound as tropomyosin blocks the muscle.
   • Cytosolic Ca2+ levels are low (stored in the sarcoplasmic reticulum).
   • Myosin heads cocked as ADP + P
2. Calcium Release & Binding to Troponin (Activation)
   • AP arrives, triggering the release of acetylcholine (ACh).
   • AP goes through T-tubules, activating the dihydropyridine receptors (DHPRs) = ryanodine receptors (RyRs) in the SR.
   • SR moves Ca2+ into the sarcoplasm.
   • Ca2+ binds troponin-C, changing the myosin-binding sites on actin.
3. Cross-Bridge Formation (Myosin-Actin Binding)
   • Myosin heads (primed with ADP + Pi) and actin form bridges.
4. Power Stroke (Force Generation)
   • Once myosin binds to actin, Pi is released, triggering the power stroke
   • Myosin causes centers of the sarcomere to move and ADP is released: low-energy state.
5. ATP Binding & Cross-Bridge Detachment
   • ATP causes myosin to be detached
6. ATP Hydrolysis & Myosin Reset (Re-Cocking)
   • ATP is hydrolyzed by myosin ATPase: re-cocking the myosin head.

Cycle

• Ends and is ready for Ca

7. Enters Relaxation (Ending the Cycle)
   • Ca goes back into SR and removes troponin.
   • The muscle rests and is awaiting stimuli