Cell Biology: Cytoskeleton Functions and Filaments

Cytoskeleton Major Functions

• Gives structure and support to cytoskeleton
• Helps organize inside cell
• Resists forces
• Network of tracks for organelle transport
• Cell contraction
• Cell movement and migration
• Beating of the cilia
• Separation of chromosomes
• Splitting parents into daughter cells during cytokinesis

3 Types of Filaments

Actin

• Plasma membrane
• Shorter and smaller than other filaments
• Drives contraction movements (like muscle)
• Binds and hydrolyzes ATP
• 2 protofilaments
• Polarized

Microtubules

• Emanating out of a centrosome
• Building blocks are alpha and beta tubules
• Cytoplasm and nucleus
• Flexible ropes resistant to stretching
• Non-polarized
• 8 tetramers form filaments
• Has lamins that organize nuclear pores and structure
• Can bind with ATP or GTP
• Formed from smaller subunits through noncovalent interactions

Intermediate Filaments

• Not discussed in detail.

Filaments: Formation and Subunits

• Protofilaments stabilize interior filaments
• Protofilaments are asymmetric and polarized (Beta at the end and Alpha at the beginning, moving upwards)

Elongation and Treadmilling

• Adding more than removing
• Add to one end and remove at the other end
• Staying the same length
• Occurs when one end is largely ADP/GDP bound and the other is largely ATP/GTP bound

Microtubule Organizing Centers (MTOCs)

• Nucleation occurs here
• Polymerization of microtubules depends on nucleation and elongation
• Beta combines GTP and hydrolyzes it into GDP

Concentrations

• Each end of a filament has a different critical concentration
• Critical concentration = rate of adding and subtracting subunits equal
• If subunit concentration exceeds critical concentration, growth occurs
• If below, disassembly occurs
• Filament formation and concentration of free subunits influence association (Kon) and dissociation (Koff) rates
• Plus end is more stable than the minus end

ATP/GTP Hydrolysis

• Controls the concentrations
• Beta binds GTP then to GDP to become a different form (D)
• Hydrolysis causes conformational changes that decrease filament affinity
• D forms are more likely to dissociate, T forms are more likely to be added

GTP Cap

• Creates a cap on plus end - making it straight and stable
• Prevents microtubule curvature that GDP subunits would have - keeping the shape round.
• Catastrophe occurs when the cap is lost.

Motor Proteins

• Convert chemical energy into mechanical energy
• Have heads that bind to filaments and tails that bind to cargo
• Binds to hydrolyze ATP
• Carries organelles to destinations
• Causes sliding of filaments

Motor Protein Types

• Myosin = actin dependent
• Kinesin = microtubule dependent, usually plus end directed
• Dynein = microtubule dependent, usually minus end directed
• Velocity = rate of physical displacement
• Processivity = how long a motor protein stays active with a filament

Regulation of Filament Formation and Polymerization

• Affected by proteins that bind free subunits
• Proteins bind monomers and expose only the plus end binding side of actin monomer, promoting plus end assembly

ARP 2/3

• Produces branched actin networks
• Acts at minus end as nucleators

Formin

• Functions at plus end of filament
• Linear, unbranched actin filaments

Thymosin

• Binds subunits to prevent assembly

Prolifin

• Binds subunits and speeds up elongation to add filaments

Regulation of Filament After Formation

• Cofilin
  • Destabilizes bound actin filaments
  • Induces a twist that loosens subunits
  • Binds to ADP actin to promote turnover of older filaments

Capping Proteins

• Stabilizes filaments
• Prevents ATP/GTP hydrolysis
• Capped on plus end after growth

Skeletal Muscles

• Made of myofibrils
• Myofibrils are made of sarcomeres
• Actin outside Z disk, myosin between
• Actin and anchoring stabilizer by capping proteins
• Nebulin binds actin
• Actin binding proteins stabilize filament
• Stays actin bound for short periods.

Sarcomere and Accessory Proteins

• Actin filaments anchor and stabilize by capping proteins
• Nebulin binds actin
• Stays actin bound for brief periods
• Actin-binding proteins stabilize filaments

Sliding Model

• Calcium is released, binds to troponin
• Moves tropomyosin, myosin connects to actin
• Myosin heads hydrolyze ATP, actin slides
• Myosin head binds to ATP and detaches from actin

Calcium Regulates Contraction

• Regulates position of tropomyosin in actin B
• By binding to move it out of the way

Flagella and Cilia

• Can propel cells through liquid media
• Flagella is long filaments causing wave movement
• Cilia are short filaments causing swimming power and recovery stroke

Axoneme

• Inside of flagella and cilia
• Connected in a circle
• Activation of dynein, motor protein arms reach and attach to neighbor and tries to walk/slide, cross-linking proteins stop gliding.

Basal Bodies

• Anchors flagella and cilia

ECM

• Network of secreted proteins and polysaccharides, forms solid substrate for cell anchoring and crawling.
• Glue that holds cells together

Collagen

• Trimers, rod-like triple helix
• Rigid fibrils, pack into thick filters
• Resistant to pulling forces

Proteoglycans

• Assembled into large complexes from core linkage
• Has complex carbohydrates

Fibronectin

• 2 polypeptides linked by disulfide bridges
• Binding sites for ECM proteins, receptors, brings together components

Laminin

• 3 Polypeptides linked by disulfide bonds
• Binds to cell surface adhesion receptors and other ECM components.

Integrins (Mediate cell-ECM attachment)

• Occurs at ECM binding sites and hemidesmosomes
• Capable of inside-out signaling (active and inactive)
• Outside-in signaling (affects cell differentiation)

Focal Adhesions

• Sites that anchor cells to substratum (ECM)
• Large clusters of integrins
• Signaling hub
• Capable of creating and responding to mechanical forces

Hemidesmosomes

• Anchor basal surface of epithelial cells to basement membrane, large clusters of integrins.
• Signaling hub
• Resist forces
• Intermediate filaments

Cadherins

• Require Ca++ for binding
• Mediate cell-cell interactions
• Catentin tethers cadherins to the cytoskeleton

Cell-Cell Anchoring Junctions

• Adherens junctions

Desmosomes

• Links intermediate filaments

Tight Junctions

• In epithelial tissues forming an impermeable barrier
• Limits lateral diffusion within the plasma membrane
• Claudins and occludins

Gap Junctions

• Allows passage of small molecules
• Link cell cytoplasms
• Connexins proteins assemble to form transcellular pores

Adhesion and Traction

• Protrusion is when cell extends membrane and attaches to substratum
• Bundles of actin and myosin contract and squeeze cell forward
• Adhesion on back breaks

FIllipodia and Lamellipodia

• Filopodia: Spike out of plasma membrane, tight bundle of unbranched actin filaments.
• Lamellipodia: ARP 3 nucleates actin at leading edge. Branched network leads to a broad flat membrane extension.

Cell Cycle

• Cells duplicate genome and divide to create more cells.
• When do cells divide? A buildup of factors hits a threshold, signaling health of the cell.
• S Phase: Replication of entire genome; starts at origin of hundreds of different located genomes.

M-phase (Mitosis)

• Spindle formation Microtubules and motor proteins Made from 2 centrosomes Separates chromosomes Astral microtubules contact cell cortex from centrosome Interpolar microtubules connect centrosomes Kinetochore attaches to chromosomes for movement.
• Chromosomal Attachment and Tension Nuclear envelope breaks down Kinetochores on chromosomes mediate attachment to microtubules Can subtract or add subunits. Kinetochore signals to loosen/tighten bonds, depending on tension levels between chromatids.
• Chromosomal Separation Microtubule disassembly and motor proteins, movement toward centrosomes Curvature of disassembly of microtubules, slides collar Strengthening dynein pulls of centrosomes toward cortex.
• Cytokinesis Occurs through actomyosin contraction, membrane insertion Microtubule spindle signals where contraction will occur. Two sister chromatids pulled in opposite directions.

Explore the major functions and types of cytoskeletal filaments, including actin, microtubules, and intermediate filaments. This quiz focuses on their roles in cellular structure, support, and movement, as well as the details of filament formation and their subunits. Test your knowledge on how the cytoskeleton contributes to cell biology.