Cellular Biology I: The Cytoskeleton

Cytoskeleton

• The cytoskeleton is a 3D network found in all eukaryotic cells.
• It's formed by microtubules, intermediate filaments, and microfilaments.
• These components are interconnected by non-covalent bonds
• The cytoskeleton structure is highly dynamic, meaning it rapidly assembles and disassembles.

The Cytoskeleton (Details)

• Microtubules:

• Polymer: αβ-tubulin heterodimers

• Diameter: Outer 25 nm, Inner 15 nm

• Functions: Organization and maintenance of cell shape, chromosome movements, intracellular transport, and cell motility.

• Microfilaments:

  • Polymer: G-actin monomers
  • Diameter: 7 nm
  • Functions: Muscle contraction, cell locomotion, cytoplasmic streaming, cytokinesis, and maintenance of animal cell shape.

• Intermediate Filaments:

  • Polymer: Various proteins
  • Diameter: 8-12 nm
  • Functions: Structural support, maintenance of cell shape, formation of nuclear lamina, and strengthening of nerve cell axons.

Functional Facts

• Mechanical and structural support
• Organelle positioning
• Movement and transport of intracellular cargo
• Cellular motility and contractility
• Cellular division

Microtubules

• Microtubules are important components of the cytoskeleton in all eukaryotic cells.
• They support the cytoplasm, form the mitotic spindle during cell division, and are central to cilia and flagella.
• Microtubules act as tracks for the movement of macromolecules and other subcellular structures.
• They are dynamic structures, capable of rapidly growing or shrinking.
• Made up of polymers of α/β tubulin
• Have a hollow tubular structure.
• Consist of 13 protofilaments.
• Polarized: Plus (+β) end and Minus (-α) end.

Microtubules: Overview

• Microtubules have extremely dynamic structures.
• They can rapidly grow or shrink depending on the number of tubulin molecules they contain.

Microtubules: Structure

• Tubular, hollow, and non-branched structures.
• Polymers of α/β tubulin.
• External diameter is 25nm, and wall thickness is 4 nm
• Wall is formed by 13 protofilaments.
• Subunits tubulin α/β heterodimers confer polarity.

Microtubule Dynamics

• Microtubules are polarized.
• Tubulin subunits are added to the plus end and removed from the minus end.
• The entire microtubule wall is built through a continuous renewal process ("treadmilling").

Microtubule Dynamic Instability

• Microtubule ends can grow or shrink at different rates.
• The rate of growth at the plus end is often faster than removal.
• The rate of growth at the minus end is slower than removal.
• Microtubule dynamics occur based on GTP-dependent dynamic equilibrium
• Microtubule growth occurs when the subunits incorporated at the end are GTP bound.

Microtubule Dynamic Instability: The Structural Cap Model

• GTP-bound tubulin dimers at the plus end form a structural cap.
• Loss of GTP from tubulin causes tension at the minus end that releases when GTP dimers are lacking.
• This process leads to "catastrophic shortening".

Microtubules' Associated Proteins (MAPs)

• Heterogeneous group of proteins.
• Take part in microtubule assembly.
• They're anchored to the microtubule wall while some extend outside.
• Form transverse bridges among microtubules.
• They contribute to multiple functions like microtubule stability or in transport mechanisms.

Microtubules' Associated Proteins (MAPs): Regulation

• The binding of MAPs to microtubules is regulated by Ser-Thr phosphorylation.
• MAP phosphorylation leads to detachment, resulting in microtubule instability and disruption.

Prolongation Neuronal

• Tau protein is an important MAP, but in Alzheimer's disease, tau proteins get overly phosphorylated causing a disruption in microtubules.

Microtubule Motor Proteins: Kinesins and Dyneins

• Convert chemical energy (ATP hydrolysis) into mechanical energy (conformational changes).
• Transport cargoes along microtubule trails.
• The cargoes include macromolecules, vesicles, mitochondria, lysosomes, chromosomes, and cytoskeletal fractions.
• Move along a unidirectional path.

Kinesins

• Superfamily of related proteins (kinesin-related proteins).
• Tetramers have heavy and light chains
• Two globular heads for binding microtubules and hydrolyzing ATP
• A neck forming a stalk, and a tail for binding cargo.
• Move along microtubules towards the plus end.
• Use a hand-over-hand mechanism.

Microtubules Motor Proteins: Dyneins

• Huge heteropolymeric protein with two heavy chains and other intermediate and light chains.
• Have a stalk and a head with microtubule-binding sites.
• The head is the force-generating engine.
• Movement is towards the minus end.

Microtubule-Organizing Centers (MTOCs)

• Structures that anchor the minus ends of microtubules.
• Centrosomes: usually adjacent to the nucleus.
• Basal bodies: below the plasma membrane.
• These structures are critical for microtubule assembly.

Centrosomes

• Main site for microtubule initiation in animal cells
• Control the number and polarity of microtubules, the number of protofilaments in their walls, and the time and location of their assembly

Basal Bodies

• Site of microtubule nucleation at the base of cilia and flagella.
• Have a similar structure to centrioles.

Microtubule Biogenesis

• Nucleation: Interaction among y- and β-tubulin. Assembly of a starting nucleus. Stabilization of bonds between subunits.
• Elongation: Addition of new subunits and rapid progressive polymerization.

Cilia and Flagella

• Structures elongating from the cell surface, having a cytoskeletal shaft (axoneme).
• Motile cilia displace cells or substances/particles and move in a perpendicular direction.
• Flagella move as a wave, pushing cells forward and move in a parallel direction.

Axoneme

• Series of parallel microtubules.
• 9 + 2 microtubule organization: nine peripheral doublets of A+B microtubules linked by interdoublet nexin bridges, and one central pair of microtubules.
• Wrapped by a sheath and connected to peripheral doublets by radial spokes.

Intraflagellar Transport (IFT)

• Motor proteins (kinesins and dyneins) for bidirectional transport along the axoneme.

Cilia and Flagellar Motility

• Motor proteins (kinesins and dyneins) mediate conformational changes that dynamically modify the angles of inclinations by causing microtubules to slide over one another

Primary Cilia

• Non-motile cellular antennae.
• Capture mechanical stimuli and transduce them inside the cell as a molecular cascade mechanotransduction
• Part of the cell signaling pathways.

The Primary Cilium

• Microtubule-based, non-motile organelle.
• Extends from basal body, receiving and processing molecular and mechanical signals.
• Discovered by Karl Zimmerman and named by Sergei Sorokin.

The Primary Cilium: Structural Facts

• Axoneme: derived from the centrosomal mother centriole; lacks the central doublet.
• Ciliary membrane continuous with the plasma membrane. Unique in composition.

The Primary Cilium: Structural Facts (Details)

• Basal body: modified centriole where the axoneme extension starts.
• Transition fibres (TF): mediate docking of the basal body to the plasma membrane.
• Transition zone (TZ): contains specialized gating structures that regulate the entrance and exit of ciliary proteins

The Primary Cilium: Structural Assembly and Maintenance

• Ciliary assembly and maintenance depend on vesicular transport from the Golgi network to the periciliary membrane.
• Vesicles are incorporated into the periciliary membrane, sometimes via lateral transport or trans-membrane diffusion.
• Intraflagellar transport (IFT) mediates the docking and fusion of vesicles on the mother centriole for axoneme extension.

The Primary Cilium: Structural Facts (Additional Details)

• Cilioplasm: a highly compartmentalized intracellular environment.
• The base of the cilium is a membrane diffusion barrier. Prevents lateral protein diffusion between plasma and ciliary membranes that are related to cell signaling, including cell proliferation, migration, and interaction with the ECM.
• Ciliary trafficking is regulated by the BBSome, a multi-protein complex.

The Primary Cilium: Functions

• A multifunctional antenna.
• Involved in apicobasal and planar cell polarity (PCP) maintenance.
• Senses mechanical/chemical changes.
• Involved in conveying signaling information to regulate cell proliferation, migration, and interactions with the ECM.
• Multiple interconnected signaling pathways like Sonic Hedgehog (SHH), Transforming Growth Factor (TGFβ), and Wnt
• Important for various cellular processes.

Primary Cilium Functions (Regulation)

• Primary cilium expression is tightly regulated during the cell cycle.
• Cilium dismantles during mitosis, and centrioles duplicate to form spindle poles
• Cilium is temporarily resorbed before S phase
• Cilium is typically lost during G0 as quiescent cells cease to sense and signal environmental cues.

The Primary Cilium: Role in Development

• During the brain's development, the integrity of the ciliary signaling is needed for:
  • Radial glia progenitor migration
  • Cortical scaffolding
  • Neuroblast orientation
  • Neuronal migration
  • Correct cortical patterning

The Primary Cilium: Role in Craniofacial Malformations

• Defects in ciliary signaling and basal body proteins cause ciliopathies.
• Ciliopathies impact different tissues and cells due to pleiotropy

Intermediate Filaments

• Fibrous, non-polarized, non-branched filaments, with a diameter of 10-12 nanometers.
• Spread throughout the cytoskeleton, providing mechanical resilience.
• Less sensitive to chemical agents than other cytoskeletal elements (more difficult to solubilize).

Intermediate Filaments: Composition

• 70 different proteins form subunits.

• Classified into 5 categories:

  • I-IV cytoplasmic
  • V nuclear

Intermediate Filaments: Structure (Tetramer)

• Basic unit: tetramer formed by two homodimers of two polypeptides.
• The a-helical rods align parallel to form a coiled coil dimer,
• Subsequently to a protofibril tetramer,
• 8 tetramers associate laterally to form larger filaments, called intermediate filaments, of~ 60 nm length.
• No chemical energy for the assembly needed.
• Important structural role to reinforce the cytoskeleton.

Intermediate Filaments: Function

• Scaffold for cellular architecture.
• Maintain cellular shape of neurons.
• Connect to the plasma membrane, microtubules, and microfilaments.
• Key to absorbing mechanical stress.

Type III Intermediate Filaments (Example)

• Vimentin, Desmin, GFAP, and Peripherin

Type IV Intermediate Filaments (Example)

• Neurofilament proteins: NF-L, NF-M, and NF-H

Type V Intermediate Filaments (Example)

• Lamin A and Lamin B form the nuclear lamina.

Microfilaments

• Composed of globular actin (G-actin) subunits.
• Polymerize into a flexible, branched filamentous actin (F-actin)
• 8 nm in diameter and polarized.
• Functions: Cellular motility, intracellular movements, phagocytosis, and cytokinesis.
• Regulated by ATP, which changes conformation in G-actin.

Microfilaments: Assembly-Disassembly

• ATP-actin monomers are added to the plus end and removed from the minus end depending on the concentration of ATP, with a critical concentration threshold.

Myosins: The Microfilaments’ Motor Protein

• Motor proteins that interact with microfilaments.
• Humans contain about 40 different myosins.
• Two general categories: Conventional (type II) myosins, found in muscle tissue, and unconventional myosins (type I and III-XVIII).

Myosins: Features

• Typically symmetrical structure/ heteroexamer: 6 polypeptides chains (heavy and light chains).
• The heads bind F-actin and have similar structure while the tails are highly divergent.

Myosin Subunits: Bipolar Filament Formation

• Myosin subunits form a bipolar filament shape.
• Tails are towards the center and heads are towards the opposite ends.
• Heads generate forces to pull two F-actin filaments toward each other, which is the basic mechanism for muscle contraction.

Muscle Contraction

• Muscle contractions are coordinated by alterations of calcium concentration with regulatory proteins like troponin and tropomyosin.

Dystrophin and DAPC

• Dystrophin: rod-shaped protein.
• Interacts with other proteins (a-dystrobrevin, syncoilin, synemin, sarcoglycans, dystroglycan, and sarcospan) to form the Dystrophin-Associated Protein Complex (DAPC)
• Links the cytoskeleton of muscle fibers to the extracellular matrix (ECM)
• N-terminal binds to F-actin, C-terminal binds to the DAPC at the sarcolemma.

Dystrophin and Muscle Dystrophy

• Dystrophin, encoded by the DMD gene, largest gene in the human genome
• Mutations lead to Muscular Dystrophies (XLR): Duchenne and Becker
• Loss/reduction of Dystrophin leads to DAPC destabilization, progressive fiber damage, and membrane leakage.
• DMD patients often become wheelchair-bound by 12 years old.

Explore the intricacies of the cytoskeleton in eukaryotic cells in this first part of the Cellular Biology course. Learn about the structure and dynamics of microtubules, intermediate filaments, and microfilaments. Engage with key concepts to enhance your understanding of cellular biology.