Cytosol & Cytoskeleton – Comprehensive Study Notes

Page 1: Cellular Organelles and Functions – Cytosol & Cytoskeleton

• Introductory slide naming the topic and lecturer (Alex R. B. Thomsen, BSc, MSc, PhD).
• Sets the scope: focus on cytosol, cytoskeleton, and especially microtubules & intermediate filaments.

Page 2: Recap – Defining the Cytosol & Cytoskeleton

• Cytosol = intracellular space between the plasma membrane and membrane-bound organelles/nucleus.
– Hosts diverse biochemical reactions, signal-transduction cascades, and bulk protein translation.
• Cytoskeleton primarily resides in the cytosol and is composed of three filament systems: actin filaments, intermediate filaments (IFs), and microtubules (MTs).
• Core cytoskeletal functions:
– Contraction (with myosin over actin).
– Determination & alteration of cell shape.
– Cell motility (crawling, chemotaxis, ciliary/flagellar beating).
– Directed intracellular transport of “cargo” (vesicles, organelles, mRNAs, proteins).
• Conceptual link: all three filament systems collaborate – e.g., vesicle budding (actin) → long-distance travel (microtubules) → short-range positioning (actin/IFs).

Page 3: Fundamental Properties of Microtubules

• Building block = tubulin heterodimer (one \alpha-tubulin + one \beta-tubulin).
– Each monomer binds GTP; only the GTP on the \beta-subunit is hydrolysable & regulates dynamics.
• Polymerisation requires GTP and proceeds head-to-tail generating a polar, hollow tube.
• Polarity:
– Plus end (+) = fast-growing, usually oriented toward plasma membrane.
– Minus end (–) = slow-growing/disassembling, tethered to a microtubule-organising centre (MTOC).
• Interact with microtubule-associated proteins (MAPs) that modulate dynamics, bundling, and interactions with other cellular structures.
• Cellular roles:
1. Vesicular/secretory cargo transport.
2. Mitotic & meiotic spindle formation/separation of chromosomes.
3. Structural backbone in cilia & flagella (axoneme) enabling motility.
4. Establishment of cell polarity & long-distance intracellular organisation (e.g., neuronal axons).

Page 4: Structural Dimensions & Diagrammatic Points

• Microtubule outer diameter \approx 24\,\text{nm}; lumen \approx 14\,\text{nm}; wall thickness \approx 8\,\text{nm}.
• Schematic emphasises:
– Minus (–) end at centrosome.
– Plus (+) end with terminal GTP-\beta-tubulin.
– Protofilaments arranged in 13 parallel tracks forming hollow tube.
• K (likely kinesin) depicted walking toward + end.

Page 5: Centrosome Architecture

1. Contains two centrioles (mother & daughter).

2. Each centriole = cylinder of 9 triplet microtubules (9×3).

3. Centrioles positioned orthogonally (≈90°).

4. Both are enveloped by pericentriolar material – protein “cloud” rich in \gamma-tubulin ring complexes acting as an MTOC that anchors MT minus ends.
   • Significance: nucleates radial MT array in interphase & bipolar spindles in mitosis; duplicates once per cell cycle (licensing step controlling division).

Page 6: Centrosome in Non-dividing Cells

• In interphase, single centrosome typically resides adjacent to nucleus; dictates overall MT polarity (minus ends inward).
• Provides tracks for secretory traffic from Golgi to periphery and positioning of organelles.

Page 7: Centrosomes as Poles of Mitotic Spindle

• Prior to mitosis, centrosome duplicates and separates to opposite cell poles.
• Each serves as spindle pole that captures kinetochores via dynamic MTs; ensures bi-orientation and equal chromosome segregation.

Page 8: Basal Bodies in Ciliated Cells

• A basal body ≅ structurally a centriole; docks at plasma membrane.
• Acts as MTOC for axoneme of each cilium.
• Conversion: centriole → basal body upon migration to cell surface (e.g., airway epithelium, oviduct, ependymal cells).

Page 9: Concept of Dynamic Instability

• MTs do not simply grow until steady state – they stochastically switch between phases of growth (polymerisation) and shrinkage (catastrophe).
• Requirements:
– Growth favoured when + end retains a protective “GTP cap” of non-hydrolysed tubulin.
– Loss of cap exposes GDP-tubulin → protofilaments curl outward → rapid depolymerisation (peeling).
• Biological rationale: allows rapid reorganisation of MT network to scout space, capture chromosomes, or redirect traffic.

Page 10–12: Molecular Sequence of Growth vs Shrinkage

(A) Growing phase
• Rate of GTP-tubulin addition > rate of GTP hydrolysis.
• Maintains stable GTP cap.
(B) Shrinking (catastrophe)
• Hydrolysis catches up → cap lost.
• GDP-tubulin weakens lateral bonds; protofilaments peel away → rapid disassembly.
• \text{Overall: } \text{growth velocity}\,\big(\upsilon+\big) \approx k\text{on}[\text{GTP-tubulin}] - k_{\text{hyd}}.
• Rescue events can restore growth if new GTP-tubulin adds before complete depolymerisation.

Page 13: Regulation of Microtubule Stability

• +-end capping MAPs (e.g., CLASP, EB1) physically block depolymerisation, converting dynamic MTs into stable scaffolds.
• Post-translational modifications (PTMs):
– Detyrosination (removal of C-terminal tyrosine on \alpha-tubulin) correlates with longevity; other PTMs = acetylation, glutamylation.
• Structural MAPs:
– High-molecular-mass MAPs (MAP1, MAP2) and Tau promote nucleation, bundling, and spacing between MTs (critical in neurons).
• Destabilising MAPs:
– Stathmin sequesters tubulin dimers & promotes curved protofilament conformation.
– Katanin severs MTs, generating new plus ends (for branching or dismantling).
• Clinical note: Tau hyper-phosphorylation → aggregation (neurofibrillary tangles) in Alzheimer’s disease.

Page 14: Microtubule-Mediated Vesicular Transport

• Long-range vesicle/organelle trafficking harnesses motor proteins that convert chemical (ATP) energy into mechanical steps along MTs.
• Directionality rule:
– Motor domain at N-terminus → walks to + end (anterograde).
– Motor domain at C-terminus → walks to – end (retrograde).
• Ensures vectorial flow: ER/Golgi → plasma membrane vs endocytic vesicles → perinuclear lysosomes.

Page 15–16: ATP-Driven Molecular Motors – Kinesin & Dynein

• Kinesins:
– Typically tetrameric (2 heavy, 2 light chains).
– “Hand-over-hand” mechanism: one head bound to MT; the other swings forward after ATP binding.
– Step size ≈ \sim 8\,\text{nm} (one tubulin dimer).
• Cytoplasmic dynein:
– Large multisubunit complex requiring dynactin adaptor for cargo linkage.
– Power-stroke generated by AAA+ ATPase ring.
• Energetics: \text{ATP} \rightarrow \text{ADP} + \text{P}_i + 50\,\text{pN·nm of work} allows 1 “step.”
• Cooperative interplay can create bidirectional “tug-of-war” motility; regulation via cargo adaptors, phosphorylation, and localized Ca^{2+}/cAMP signals.

Page 17–18: Visual Animations (Linked Videos)

• The cited YouTube resources provide dynamic 3D depictions of walking motors and vesicle traffic; recommended for reinforcing spatiotemporal understanding.

Page 19: Organelle Movement & Spatial Organisation

• MT array defines trafficking axes:
– Golgi typically localises near minus-end-rich centrosome.
– ER network extends toward plus ends.
• Perturbation (e.g., nocodazole) collapses Golgi and impairs secretion; underscores MT importance for organelle homeostasis.

Page 20–23: Cilia – Structure & Physiological Role

• Motile cilia line trachea, bronchi, uterine tubes; beat to propel fluids/particles (mucociliary clearance).
• Beating is cyclical:
– Power stroke (effective stroke) moves fluid.
– Recovery stroke resets without reversing flow (asymmetric waveform).
• Defects (primary ciliary dyskinesia) → chronic respiratory infections, infertility, situs inversus (due to nodal cilia failure).

Page 24: Axonemal 9 + 2 Microtubule Array

• Core of motile cilium/flagellum: 9 outer MT doublets + 2 central singlets (9 + 2).
• Accessory structures:
– Outer & inner dynein arms (generate force).
– Nexin links (elastic connectors).
– Radial spokes + central sheath (regulate dynein activity).
• Diameter ≈ 100\,\text{nm}; ensheathed by specialised ciliary membrane.

Page 25–26: Mechanism of Bending vs Sliding

(A) Isolated MT doublets + ATP → dynein “walks” causing sliding.
(B) In intact axoneme, sliding constrained by nexin → converted to bending; sequential activation on opposite sides creates waveform.
• Coordination hypothesised to involve Ca^{2+}, phosphorylation, and central pair rotation.

Page 27: Integrated Animation (External Link)

• 3D animation shows alternating activation/inhibition of dynein arms orchestrating ciliary/flagellar motion.

Page 28: Intermediate Filaments – Overview

• IFs = heterogeneous fibrous proteins (keratins, vimentin, desmin, neurofilaments, lamins).
• Assembly is spontaneous, energy-independent; subunits form parallel coiled-coil dimers → staggered antiparallel tetramers → protofilaments → \approx 10\,\text{nm} filaments.
• Non-polar (no +/– ends), no motor proteins; primarily confer tensile strength.

Page 29: Cytoplasmic & Nuclear IF Classes

• Cytoplasmic:
– Keratins (epithelia, hair, nails).
– Vimentin & vimentin-like (mesenchymal cells, muscle, glia).
– Neurofilaments (neurons).
• Nuclear: Lamins (form nuclear lamina underlying inner nuclear membrane; disassemble during mitosis via phosphorylation by Cdk1).

Page 30: Molecular Architecture of IFs

A–F panels (slide):
A. \alpha-helical rod domain shared by all IF proteins.
B. Coiled-coil dimer (parallel).
C. Staggered tetramer (antiparallel, non-polar).
D–F. Eight tetramers laterally associate → protofilament; four protofilaments wrap to final 10\,\text{nm} filament.
• Axial repeat ≈ 21\,\text{nm}.
• Mechanical property: can be stretched ~250% without breaking (hyper-extensible contrast to MTs/actin).

Page 31: Mechanical Resilience – Stretching Experiment

• Epithelial sheets with intact IF network withstand mechanical stretching; removal of IFs causes rupture (“cells remain intact and together” caption).
• Demonstrates role in tissue integrity under shear or tensile stress.

Page 32: IFs in Cell–Cell & Cell–Matrix Junctions

• Desmosomes (cell–cell) & hemidesmosomes (cell–ECM) anchor keratin IFs.
• Skin layers: Epidermis (keratinocytes linked by desmosomes) vs Dermis; IF anchorage prevents blistering.
• Pathology: mutations in keratin → epidermolysis bullosa simplex (fragile skin).

Page 33: Concluding Summary (Slide)

• Microtubules: polar, GTP-regulated, anchored at centrosomes (– end), dynamic instability at + end. Roles in division, transport, motility.
• Intermediate filaments: diverse proteins, non-polar, energy-independent assembly, provide mechanical stability at cellular & tissue levels.
• Integrative thought: cytoskeletal crosstalk ensures cells are both “hard-wired” (IFs) and “re-programmable” (dynamic MTs & actin) to adapt to developmental cues, mechanical stress, and environmental signals.