Cell Biology- Chapter 17

Intermediate Filaments and Microtubules

The cytoskeleton is composed of three classes of fibers

  • Intermediate filaments

  • Microtubules

  • Actins

Intermediate filaments form a strong network within the between cells

  • composed of twisted helical fibers

  • There are distinct cytoplasmic and nuclear intermediate filaments: Keratin, vimentin and vimentin-related filaments, neurofilaments, nuclear lamins

Keratin mutations reduce integrity of the skin

  • keratin forms a network that links adjacent skin cells to each other and the basal lamina (via hemidesmosomes)

  • Mutations that decrease the strength of keratin networks results in epidermolysis bullosa, a skin disorder that causes blisters, resulting from the epidermis separating from dermis.

The nuclear lamina is composed of lamin intermediate filaments

  • lamin proteins assemble in a mesh-like network under the nuclear envelope

  • During mitosis, phosphorylation of lamin proteins weakens the network and leads to disassembly of the nuclear envelope. after mitosis, the lamins are dephosphorylated, which facilitates nuclei formation.

  • Lamin A mutations disrupt nucleus structure (example: progeria)

Plectin links intermediate filaments to other cytoskeletal proteins

  • the strength of cytoskeletal networks is enhanced by linkages between different fiber types

  • plectin isn’t needed to establish intermediate filaments networks, but is essential to stabilizing them- inactivating plectin results in devastating disorders.

Envelope-spanning protein complexes link the nuclear lamina to the cytoskeleton

  • complexes that span the nuclear envelope likewise link the nuclear lamina and chromatin to elements in the cytosol

  • Trans-envelope interactions mediated by KASH and SUN domains

Microtubules expand from an organizing center and contribute to diverse functions

  • microtubules have distinct polarity, + and - ends and originate from organizing centers

  • Centrosomes are located near the nucleus and are anchor points for the mitotic spindle, Microtubules cilia cores extend from basal bodies

Microtubules are hollow tubes composed of linear dimer strands

  • a microtubule is produced of alpha and beta tubulin dimers

  • alpha-beta tubulin dimers form linear protofilaments

  • the orientation of the dimers is consistent, alpha tubulin points to - end, beta tubulin points to + end

  • because microtubules extend via addition of dimers, their polar nature is maintained

  • Microtubules both gain and lose dimers at + end, the - end is more stable.

  • Microtubule polarity allows form directed movement of tubulin-binding motor proteins

Microtubules polymerize from nucleation sites on the centrosome

  • a centrosome consists of a pair of centrioles, a diffuse matrix, gamma tubulin, and the - end of microtubules

  • gamma-tubulin facilitates nucleation of the first tubulin dimers of the new microtubule.

Each microtubule grows and shrinks independently of it’s neighbors

  • microtubules have dynamic instability, unless stabilized, a microtubule will grow and shrink at its + end

Microtubule stability is influenced by GTP/GDP

  • have the binding sites

  • GTP-tubulin dimers have higher affinity for each other than GDP dimers

  • GTP hydrolysis by tubulin is slow

  • As long as fresh GTP tubulin dimers are available, a microtubule will grow rapidly.

  • If GTP- tubulin dimers are not available, or if GTPase activity is accelerated, the GTP cap can be lost

  • microtubule catastrophe: rapid shortening when GDP dimers readily leave the tubule.

Microtubule capping proteins stabilize ends and support directional arrangements

  • the centrosome is constantly shooting out

  • If the plus end encounters a stabilizing GTP capping protein, it doesn’t retract

  • stabilizing proteins throughout the cell…. (need more notes)

Microtubule binding motor proteins transport vesicles and organelles

  • The polarized structure of microtubules allows for directed movement of motor proteins

  • Different motor proteins have towards the + or - end …. (need more notes)

Dynein and Kinesin motor proteins being microtubules with globular heads

  • Kinesin walks toward + end

  • Dynein walks toward - end

Different motor proteins carry different cargos and move in different directions

  • different motor proteins have different cargo- some bind directly, others bind with adaptor proteins

  • in general, kinesins are + end directed, and dyneins are - end directed, there are exceptions though

  • motor proteins deliver molecules, vesicles, and whole organelles

Microtubule motor proteins position organelles

  • plus-end directed motors spread ER throughout the microtubule network

  • conversely, minus end directed motors keep the Golgi close to centrosome and nucleus

Motile cilia have microtubule cores

  • when motor proteins are immobile, they have microtubules

  • motile cilia have microtubule cores that bend stiffly with the actions of motor proteins and whip back elastically when motor proteins release.

Wave-like movement of a flagellum propels a spermatozoon

  • animal spermatozoa are propelled by a cilium called a flagellum

  • cilia and flagella share a structure of 9 microtubule doublets surrounding a pair of singlet microtubules (9+2 is highly conserved)

  • Immobilized ciliary dynein motors produce controlled bending motions that move the structure.

Motor proteins make free microtubules

  • in cilia and flagella, the tubules are linked to each other, so they bend instead of slide

  • timed bending produces a whip-like motion down flagella that provide propulsion to the spermatozoan

  • Kartagener’s is a disorder of non-functional ciliary dynein. These cilia are crucial for moving mucus, bacteria, and debris out of the lungs and airways. In Kartagener's syndrome, the cilia don't function properly, leading to various health issues12.


Actin Filaments and Muscle Contraction

Actin microfilaments support cell shapes

  • in contrast to cilia, microvilli have cores of actin

  • contractile bundles act like small muscles that control the overall shape and size of cell

  • Filopodia are thread-like projections associated with cell movement

  • contractile rings form cleavage furrow that separates daughter cells at the conclusion of cytokinesis

Microfilaments are thin fibers composed of monomeric subunits

  • composed of actin protein chains

  • non-polymerized actin monomers are globular (g-actin). polymerized actin is called filamentous (f-actin)

  • Unlike microtubules, f-actin is composed of monomers

  • Each actin monomer is asymmetric with a barbed end and a pointed end

  • f-actin is polar with a plus end and a minus end

Monomers are most frequently added at the + end and lost at the - end

  • actin monomers have a greater affinity for each other when bound to ATP

  • At intermediate actin concentrations, ATP actin joins at the + end and ADP-actin leaves at the - end (treadmilling)

Actin-binding proteins control the behavior and structure of actin filaments

1. Nucleating Proteins

These proteins help initiate the formation of actin filaments. Examples include the Arp2/3 complex and formin proteins1.

2. Severing and Capping Proteins

These proteins regulate the length of actin filaments by severing them or capping their ends. Gelsolin and cofilin are well-known severing proteins, while CapZ is a capping protein1.

3. Crosslinking Proteins

These proteins organize actin filaments into networks or bundles. Filamin and α-actinin are examples of crosslinking proteins1.

4. Motor Proteins

These proteins use ATP to move along actin filaments, transporting cellular cargo. Myosin is the most prominent motor protein associated with actin1.

5. Binding and Stabilizing Proteins

These proteins bind to actin filaments and stabilize them. Tropomyosin is a key stabilizing protein that binds along the length of actin filaments1.

6. Regulatory Proteins

These proteins control the dynamics of actin polymerization and depolymerization. Profilin and thymosin β4 are examples of regulatory proteins1.

7. Linking Proteins

These proteins link actin filaments to other cellular structures, such as membranes. Dystrophin and spectrin are examples of linking proteins1.

Forces produced by actin-rich cortex assists in cell movement

  • cells that lack motile organelles move by crawling around substrate

  • 1. Actin Polymerization

    At the leading edge of the cell, actin filaments rapidly polymerize, pushing the cell membrane forward to form structures called lamellipodia and filopodia1. This polymerization creates a protrusive force that extends the cell's front.

    2. Adhesion Formation

    As the cell membrane is pushed forward, the cell forms focal adhesions with the substrate. These adhesions anchor the cell to the surface, providing traction for movement1.

    3. Actin-Myosin Contraction

    At the rear of the cell, myosin II motor proteins interact with actin filaments to generate contractile forces. This contraction pulls the cell body forward, allowing the cell to move1.

    4. Coordination of Actin Networks

    The actin cortex coordinates the activities of polymerizing and contracting actin networks. Actin arcs at the cell's lamella provide a base against which the lamellipodium's polymerizing actin filaments protrude1. This interplay between actin arcs and focal adhesions creates a stiff substrate for effective crawling.

    5. Dynamic Remodeling

    Throughout this process, the actin cytoskeleton undergoes continuous remodeling, allowing the cell to adapt its shape and movement in response to environmental cues1.

    This dynamic and coordinated activity of the actin cortex enables cells to crawl efficiently on various substrates, playing crucial roles in processes like wound healing, immune response, and tissue development.

  • Lamellipodia- “sheet feet”

  • Filopodia- “thread feet”

Branching actin networks extend into the leading edge of a migrating cell

  • branching proteins bundling F- actin into a dense network at the leading edge

  • Rapid plus end elongation of multiple filaments press the membrane outwardly

Activation of different Rho family small G proteins affects actin differently

  • The Rho family of small G proteins, including RhoA, Rac1, and Cdc42, each uniquely influence actin filament dynamics through distinct signaling pathways and interactions with specific effector proteins. Here's a more detailed look at how each of these proteins affects actin differently:

  • RhoA

Effect on Actin: Promotes the formation of stress fibers and focal adhesions.

Mechanism: RhoA activates Rho-associated protein kinase (ROCK), which phosphorylates myosin light chain (MLC) and inhibits myosin light chain phosphatase (MLCP). This increases actin-myosin contractility, leading to the assembly of stress fibers.

  • Rac1

Effect on Actin: Induces the formation of lamellipodia, which are broad, sheet-like protrusions at the leading edge of migrating cells.

Mechanism: Rac1 activates the WAVE complex, which subsequently activates the Arp2/3 complex. This promotes actin nucleation and branching, resulting in a dense network of actin filaments in lamellipodia.

  • Cdc42

Effect on Actin: Promotes the formation of filopodia, which are slender, finger-like protrusions.

Mechanism: Cdc42 activates the WASP family of proteins, which also activate the Arp2/3 complex. This leads to the nucleation of actin filaments and the formation of parallel bundles of actin in filopodia12.

  • Why Different Effects?

The distinct effects of Rho family G proteins on actin filaments arise from their ability to interact with specific sets of effector proteins that regulate actin dynamics. These interactions activate different signaling pathways, which control various aspects of actin filament organization and behavior. This specialization allows cells to precisely control their shape, movement, and adhesion in response to various external signals, enabling them to perform a wide range of functions.

Myosin-I is a one headed motor that moves toward the plus end of the chain

  • ATP powered conformational changes allow myosin-I to transport vesicles towards + end of the f-actin

  • can slide actin fibers

Myosin II bundles power muscle contraction

  • a single myosin II has a helical dimerization domain that allows multiple proteins to bundle together

  • A myosin-II filament (thick filament) consists of many staggered proteins -heads are oriented in opposite directions

A muscle cell contains multiple myofibrils (sarcomere bundles)

  • skeletal muscle cells are long tubes that originate from a fusion of precursor cells

  • most of a muscle cell is filled with myofibrils- nuclei are pushed out to the periphery of the cell

Sarcomeres

  • composed of thick filaments (mysosin II) , thin filaments (Mysosin I)

  • need more notes

Myosin II has a cross-bridging cycle

  • Attachment: Myosin heads bind to actin filaments, forming a cross-bridge. (think rigor mortis)

  • Power Stroke: The myosin head pivots, pulling the actin filament toward the center of the sarcomere. This movement is powered by the hydrolysis of ATP.

  • Detachment: A new ATP molecule binds to the myosin head, causing it to detach from the actin filament.

  • Reactivation: The ATP is hydrolyzed to ADP and inorganic phosphate (Pi), re-cocking the myosin head to its original position, ready to form another cross-bridge.

Transverse tubules are sarcoplasmic reticulum surround myofibrils

  • T-tubes: T-tubules are invaginations of the muscle cell membrane (sarcolemma) that penetrate into the cell's interior. They play a crucial role in the excitation-contraction coupling process.

    Functions of T-tubules

    1. Action Potential Transmission: T-tubules help transmit the action potential from the sarcolemma deep into the muscle fiber, ensuring that the signal reaches all parts of the muscle cell simultaneously.

    2. Calcium Release: The action potential in the T-tubules triggers the release of calcium ions (Ca²⁺) from the sarcoplasmic reticulum (SR), which is essential for muscle contraction.

Action potentials open voltage-gated calcium channels

  • calcium triggers uncovering of myosin-II binding sites on the thin filaments

Skeletal muscle contraction- from neurotransmitter to crossbridge cycling