mol bio lecture 6.pdf

1.Compare and contrast actin filaments, microtubules, and intermediate filaments in terms of each of the following: structure, role in cell shape or organization, role in cell motility or migration, mechanisms of assembly, and mediators involved in assembly Cytoskeleton - 3 parts: Microtubule...

1.Compare and contrast actin filaments, microtubules, and intermediate filaments in terms of each of the following: structure, role in cell shape or organization, role in cell motility or migration, mechanisms of assembly, and mediators involved in assembly Cytoskeleton - 3 parts: Microtubules, Actin, Intermediate filaments - Structure: rigid but flexible, structure, shape, and support of cell - Function: movement, migration, gastrulation, spindle/contractile ring formation, reorganization, Structure Actin Filaments: microfilaments, two chains (helix), actin and myosin Microtubules: major component, Hollow, dimer composed of α- and β-tubulin. usually anchored at the centrosome. Intermediate Filaments: most diverse, thick,made of various proteins, such as keratins, vimentin, or neurofilaments. do not have inherent polarity. Role in Cell Shape or Organization Actin Filaments: Provide mechanical support and maintain cell shape, cell division (cytokinesis), muscle contraction Microtubules: Organize the cell by positioning organelles and forming the mitotic spindle during cell division. provide tracks for vesicular transport. Post-translational modification of tubulin critical for proper cell formation. Intermediate Filaments: add strength, bear tension, reinforce cell shape. Role in Cell Motility or Migration Actin Filaments: Play a central role in cell motility by forming structures like lamellipodia, filopodia, and stress fibers, allowing cells to move or change shape. Actin-myosin interactions generate contractile forces. Microtubules: ○ Vesicular trafficking Kinesin: Anterograde transport (to membrane, - end) Dynein Retrograde transport (from membrane, + end) ○ Positioning of the Centrosome Aids in directed cell migration. ○ Cilia and Flagella Cell motility Intermediate Filaments: Do not directly participate in motility but provide fixed positions of organelles and have insoluble fibers. Mechanisms of Assembly  Actin Filaments: Assembly is ATP-dependent. growth/assembly requires ATP. hydrolysis of ATP shortens fibers. Polymerization and branching of actin via nucleation. Microtubules: Assembly is GTP-dependent. Bind GTP to promote growth. GTP-hydrolysis frees dimers. Mediators Involved in Assembly Actin Filaments: Mediated by actin nucleators such as ARP2/3 complex and NPFs for polymerization. Tension and forces cause reorganization. Microtubules: Assembly is mediated by patterns of TubA and TubB isoforms 2.Explain the mechanisms through which a basic heterodimer can gain increased variation using microtubules and tubulin isoforms as an example ◦Summarize the role of tubulin subtypes in tubulin structure and function within different cell populations ◦Link the tubulin code with how tubulin components influence microtubule behavior, dynamics, binding, and function Mechanisms for Increased Variation in a Heterodimer Using Microtubules and Tubulin Isoforms as an Example Tubulin, the fundamental building block of microtubules, exists primarily as a heterodimer consisting of α- and β-tubulin. Despite the simplicity of this dimeric structure, variation is introduced through: 1. Multiple Tubulin Isoforms: Both α- and β-tubulin have multiple gene-encoded isoforms, each differing slightly in amino acid sequence. Build & Break to move chromosomes. 2. Post-translational Modifications (PTMs): Tubulin proteins undergo various PTMs. These modifications increase heterogeneity and regulate microtubule behavior, dynamics, and interactions with other proteins. 3. Mutations in Tubulin Isoforms: changes in depolymerization and activity affects growth and motility. Tubulinopathies in TubB isoforms can cause brain malformations. GTP binding affects dynamics, reducing disassembly impacts overall function 3.Identify the actin arrangements generated within cells and summarize their role in conferring cell geometry and the generation of force in both cell geometry and migration 1. Microtubule Organization and Role in Tissue remodeling PTM of tubulin: critical for proper cell formation Variable organization based on cell type: radial vs non radial arrays ○ Radial protrusion: dividing cells, proliferation, centrosome attachment  ○ Non-Radial arrays Non-dividing, differentiated cells Epithelial cells, neurons, cilium, platelet, fibroblast 1. Actin structures Branched and Bundled Actin ○ Resist compressive force from cells ○ Exerts protrusive force on cells Cortical Networks ○ Exert tension in all directions Stress fibers ○ Arrangement: Contractile bundles of actin filaments arranged in antiparallel arrays, associated with myosin II. ○ Role in Cell Geometry: Stress fibers provide rigidity and structural integrity, particularly in elongated or spread-out cells. They also connect to focal adhesions, anchoring the cell to the extracellular matrix. ○ Role in Force Generation: Stress fibers generate contractile forces through actin-myosin interactions. These forces are critical for cell migration and maintaining tension at focal adhesions, allowing the cell to move by contracting the rear and pulling itself forward. ○ Role in Cell Geometry: The actin cortex provides mechanical support to the cell's shape and defines the cell's overall geometry. It enables cells to withstand tension and resist deformation. Role in Force Generation: By regulating cortical tension, the actin cortex influences the cell’s ability to change shape during processes like cytokinesis or shape remodeling during migration. Actin arrangements: essential for maintaining and altering the structural and functional dynamics of cells during processes like migration, cytokinesis, and morphogenesis. 2. Microtubules and Actin In Epithelial Morphogensis Arrangement: Microtubules play a role in cell shape changes and cell mechanics Role in Cell Geometry: actin and myosin form contractile arrays that are essential to processes of morphogenesis. Morphogenesis in Drosophila: both actin and microtubules ○ Epithelial folding: Key to shaping organs and tissues ○ Epithelial Elongation: Remodeling within the plane of the tissue Actinopathy-Associated Protrusion Effects - Uropod,Filopodia, lamellipodium, podosomes, microvilli 1. Uropod Description: The trailing end of a migrating cell, particularly in leukocytes and other motile cells. Role: Helps retract and guide the rear of the cell during migration, maintaining polarity and directionality by coordinating with the leading edge. 2. Filopodia Description: Thin, finger-like projections composed of tightly bundled actin filaments. Role: Act as sensory structures that probe the extracellular environment, guiding cell migration by sensing chemical and mechanical signals. 3. Lamellipodium Description: Broad, flat, sheet-like protrusions generated by dense networks of branched actin filaments. Role: Forms the leading edge of migrating cells, pushing the membrane forward and driving cell movement. 4. Podosomes Description: Actin-rich, dot-like structures associated with the plasma membrane. Role: Involved in adhesion, mechanosensing, and matrix degradation, commonly found in cells involved in tissue invasion, such as macrophages and osteoclasts. 5. Microvilli Description: Finger-like extensions of the plasma membrane, supported by parallel bundles of actin filaments. Role: Increase surface area for absorption in epithelial cells, particularly in the intestines, and play a role in maintaining cell polarity and signaling. Summary of Roles in Cell Geometry and Migration Cell Geometry: Actin structures like the cortex, lamellipodia, and stress fibers shape the cell and control its deformation and elasticity. Force Generation: Actin polymerization in lamellipodia and filopodia provides the protrusive forces needed for migration, while contractile forces in stress fibers and the contractile ring allow for cell movement and division. These forces ensure that cells can effectively change shape, move, and divide in response to environmental and intracellular signals. 4.Summarize the steps involved in the formation of cellular protrusions such as lamellipodia and focal adhesions ◦Outline the key features, formation, and function of each of the following: protrusions, cellular polarity, and focal adhesions. Cell Migration - Dynamic process Steps of mesenchymal migration: 1. Activation of signal pathway: external signals 2. Nucleation of Actin: Arp2/3 3. Cell polarization: Direction 4. Frontal protrusions: fight compression a. Example: Lamellipodium 5. Focal adhesions: generate contractile forces 6. Cell body contracts 7. Mature FA dissemble from rear and rear retracts: movement generation Key Features, Formation, and Function of Cellular Protrusions, Polarity, and Focal Adhesions 1. Protrusions Key Features: Actin-rich extensions, 2 types (lamellipodia, filopodia) have distinct regions of actin and microtubules. Extension of components is TOWARDS the cell periphery. Formation: Initiated by the nucleation and polymerization of actin filaments, driven by signaling pathways involving Arp2/3 complex. Increased level of protease activity to destroy proteins allowing for rearrangement Function: Protrusions drive cell migration, allowing the cell to explore its environment, interact with the extracellular matrix, and move toward signaling cues. required to build and break arrangements. 2. Cellular Polarity Key Features: polarized cytoskeletal networks form in the same direction of movement. Formation: Actin fibers orient and direct microtubules using crosslinking protein ACF7. Cellular polarity is established through signaling pathways (Rac and Rho) that regulate the cytoskeleton, membrane trafficking, and focal adhesion dynamics. Function: Cellular polarity ensures directional movement and enables the cell to maintain forward migration 3. Focal Adhesions  Key Features: Large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix via stress fiber-linked focal adhesions. mature and strengthen in response to tension. Formation: Focal adhesions form at sites where integrins bind to matrix proteins and recruit proteins which connect to actin filaments. APC facilitates association between actin and tubules. Rho-GTPases play critical roles in adhesion and protrusion during migration via focal adhesion maturation. Function: Focal adhesions provide traction for cell migration, transmit mechanical signals, and anchor the cell to the extracellular matrix, stabilizing protrusions and guiding movement. 5.Evaluate the cross-talk between cytoskeleton components in cell motility and migration and assess how changes in cytoskeletal components and associated molecules impact cell geometry, motility, and migration 1. 4 Forces of Migration in Embryogenesis Polarization - Gives directionality to the movement - Defines front from back - Remodeling within a cell Protrusion - Actual start of movement/migration - Involves leading edge and polymerization of actin creating protrusive forces Adhesion - Attachment to surrounding matrix via integrins and formation of focal adhesions - Gives cell pushing off force Retraction - Release of adhesions at the rear to enable forward movement 2. Cytoskeleton rearranges to fight the 4 forces - Cellular protrusions - have distinct regions of actin and microtubule enrichment - Cell type specific patterns - of actin and microtubules have been observed both in the cell structure and in the development of protrusions including lamellipodia - Focal adhesions - play a key role in anchoring fibers required to produce contractile stress forces 6.Explain the consequences of changes in expression and primary protein structure for microtubules and actin filaments in terms of activity and human disease (causes of tubulinopathies and actinopathies) Lissencephaly - Heterogeneous groups of disorders characterized by loss of normal gyral patterns - Causes: - Abnormal neuronal migration - Defects in tubulins - Symptoms/Effects - Psychomotor impairment - Agyria (lack of brain fold) Actin Disorders - Cardiac, skeletal, and smooth muscle contain alpha-actin - ACTA1 = skeletal muscle alpha-actin - ACTA2 = smooth muscle alpha-actin - ACTC1 = cardiac alpha-actin