Unit 3: Cytoskeleton and Nucleus - Subjective Questions

The cytoskeleton is a dynamic network of protein filaments distributed throughout the cytoplasm and, in some cases, associated with the nucleus. It consists mainly of microtubules, intermediate filaments, and actin filaments.

Major functions:

• Cell shape and mechanical support: It maintains cell shape and resists deformation.
• Intracellular transport: Microtubules and actin filaments provide tracks for motor proteins that move vesicles and organelles.
• Cell movement: Actin filaments participate in cell crawling, while microtubules help organize cilia and flagella.
• Cell division: Cytoskeletal elements form the mitotic spindle and contractile ring.
• Organelle positioning: The cytoskeleton helps distribute and anchor organelles within the cytoplasm.
• Cell signaling: Cytoskeletal rearrangements transmit and respond to signals from the extracellular environment.
• Nuclear organization: Cytoskeletal networks interact with the nuclear envelope and help position the nucleus.

Structure: Microtubules are hollow, cylindrical polymers approximately 25 nm in diameter. Their walls are composed of 13 longitudinal protofilaments, each made of repeating alpha-tubulin and beta-tubulin heterodimers.

Polarity:

• The plus end generally grows and shrinks more rapidly.
• The minus end is often anchored at a microtubule-organizing center, such as the centrosome.
• Tubulin dimers are arranged in a uniform orientation, giving the microtubule structural polarity.

Functions:

• Maintain cell shape and resist compression.
• Form tracks for kinesin- and dynein-dependent transport.
• Organize the mitotic spindle during cell division.
• Form the core of cilia and flagella.
• Position organelles such as the Golgi apparatus and endoplasmic reticulum.
• Contribute to cell polarity and directional movement.

Microtubules undergo dynamic instability, a process in which individual microtubules alternate between phases of growth and shortening.

• Tubulin dimers bind GTP before being incorporated into a growing microtubule.
• After incorporation, the GTP bound to beta-tubulin is hydrolyzed to GDP.
• A growing microtubule has a stabilizing GTP cap at its plus end.
• If GTP-tubulin addition is faster than GTP hydrolysis, the microtubule continues to grow.
• If the GTP cap is lost, the protofilaments curve outward and rapid depolymerization, called catastrophe, occurs.
• Regrowth after shortening is called rescue.

This behavior allows cells to rapidly reorganize their microtubule network in response to changes in cell shape, intracellular transport requirements, and cell division.

Intermediate filaments are rope-like cytoskeletal fibers with a diameter of approximately 10 nm. They are formed by the assembly of fibrous protein subunits into dimers, tetramers, and finally mature filaments.

Structural features:

• They are more stable and less dynamic than microtubules and actin filaments.
• Their subunits have a central rod-shaped domain and variable head and tail regions.
• They do not have a plus or minus end and therefore lack structural polarity.

Functions:

• Provide tensile strength and resist mechanical stress.
• Maintain the structural integrity of cells and tissues.
• Anchor organelles, including the nucleus.
• Strengthen cell junctions such as desmosomes and hemidesmosomes.
• Support the nuclear envelope through the nuclear lamina.

Examples include keratins in epithelial cells, vimentin in mesenchymal cells, neurofilaments in neurons, and lamins in the nucleus.

Together, these systems form an integrated cytoskeletal framework. Microtubules mainly organize long-range transport, intermediate filaments provide mechanical stability, and actin filaments support changes in cell shape and contractile activity.

Actin filaments, also called microfilaments, are approximately 7 nm in diameter and are composed of globular actin subunits called G-actin. These subunits polymerize to form filamentous actin, or F-actin.

Assembly:

• G-actin binds ATP and associates to form a small nucleus.
• Additional actin subunits are added to both ends of the filament.
• The plus end usually grows more rapidly than the minus end.
• ATP is hydrolyzed after polymerization, influencing filament stability and turnover.

Functions:

• Form the cell cortex beneath the plasma membrane.
• Maintain cell shape and produce membrane protrusions.
• Participate in cell crawling through lamellipodia and filopodia.
• Form the contractile ring during cytokinesis.
• Interact with myosin during muscle contraction and other cellular movements.
• Support microvilli and contribute to endocytosis and intracellular trafficking.

Actin filaments interact with myosin motor proteins to generate force and movement.

• Myosin has motor heads that bind to actin and hydrolyze ATP.
• ATP hydrolysis changes the conformation of the myosin head.
• The myosin head attaches to an actin filament, performs a power stroke, and moves relative to the filament.
• Repeated cycles of attachment and detachment produce sliding between actin and myosin.
• In muscle cells, organized actin and myosin arrays produce contraction.
• In non-muscle cells, actomyosin networks control cell shape, cell adhesion, cytokinesis, and migration.
• During cell crawling, actin polymerization pushes the plasma membrane forward, while myosin contraction helps pull the cell body toward the leading edge.

Thus, actin polymerization and myosin-based contraction work together to generate both protrusive and contractile forces.

The nuclear envelope is a double membrane system that surrounds the nucleus.

• The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum and may contain ribosomes.
• The inner nuclear membrane contains specific proteins that bind nuclear lamins and chromatin.
• The perinuclear space lies between the two membranes and is continuous with the lumen of the endoplasmic reticulum.
• Nuclear pore complexes span both membranes and provide controlled communication between the nucleus and cytoplasm.
• The outer nuclear membrane can be continuous with the endoplasmic reticulum, allowing the nuclear envelope to function as a specialized region of the endomembrane system.
• The nuclear envelope breaks down and re-forms during the cell cycle in many eukaryotic cells.

This arrangement separates nuclear processes, such as transcription and DNA replication, from most cytoplasmic activities while still permitting regulated exchange.

The nuclear pore complex, or NPC, is a large protein assembly embedded in the nuclear envelope. It forms a selective channel through which molecules move between the nucleus and cytoplasm.

Structural components:

• It has an eightfold symmetrical arrangement around a central transport channel.
• Ring structures are associated with the cytoplasmic and nuclear surfaces.
• A central scaffold supports the pore and connects the two nuclear membranes.
• Filaments extend into the cytoplasm.
• A basket-like structure projects into the nucleoplasm.
• Flexible nucleoporins containing phenylalanine-glycine, or FG, repeats form the selective permeability barrier.

Transport control:

• Small ions and molecules can diffuse through the NPC.
• Large proteins and RNA-protein complexes require transport receptors.
• Importins and exportins recognize specific transport signals.
• The direction of transport is controlled by the Ran GTPase system.

Thus, the NPC is both a physical channel and a molecular checkpoint.

The nuclear lamina is a dense meshwork of intermediate filament proteins lining the inner surface of the inner nuclear membrane.

Composition and location:

• It is mainly composed of type V intermediate filament proteins called nuclear lamins.
• The lamina is attached to inner nuclear membrane proteins.
• It is connected to nuclear pore complexes, chromatin, and the cytoskeleton through nuclear envelope proteins.

Functions:

• Provides mechanical support to the nuclear envelope.
• Maintains nuclear shape and prevents deformation.
• Helps organize nuclear pore complexes.
• Anchors regions of chromatin at the nuclear periphery.
• Contributes to DNA replication, transcriptional regulation, and chromosome organization.
• Participates in nuclear envelope breakdown and reassembly during cell division.
• Helps connect the nucleus to the cytoskeleton through linker protein complexes.

Defects in lamins or associated proteins can cause nuclear deformation and are linked to several human diseases called laminopathies.

Nuclear lamins perform both structural and regulatory functions.

Maintenance of nuclear integrity:

• Lamin filaments form a supportive meshwork beneath the inner nuclear membrane.
• They distribute mechanical forces across the nuclear surface.
• They connect the nuclear envelope to the cytoskeleton and help position the nucleus.
• They stabilize nuclear pore complexes and membrane proteins.

Regulation of gene expression:

• Lamin-associated proteins attach specific regions of chromatin to the nuclear periphery.
• These peripheral chromatin regions are often transcriptionally inactive and enriched in heterochromatin.
• By controlling the spatial organization of chromatin, lamins influence which genes are accessible to transcription factors.
• Lamin interactions also affect DNA replication, repair, and chromosome organization.

Therefore, the nuclear lamina is not merely a structural scaffold; it is also an important regulator of nuclear organization and cellular gene expression.

Nuclear protein import is a selective, receptor-mediated process.

1. A protein destined for the nucleus contains a nuclear localization signal, or NLS, commonly enriched in basic amino acids.
2. The NLS is recognized by an import receptor, usually an importin complex.
3. The importin-cargo complex binds to FG-repeat nucleoporins and moves through the selective barrier of the nuclear pore complex.
4. In the nucleoplasm, Ran-GTP binds to the import receptor.
5. This binding causes the receptor to release its cargo inside the nucleus.
6. The import receptor associated with Ran-GTP returns to the cytoplasm.
7. Cytoplasmic GTP hydrolysis converts Ran-GTP to Ran-GDP and releases the receptor for another transport cycle.

The process is directional because Ran-GTP is concentrated in the nucleus, whereas Ran-GDP is concentrated in the cytoplasm.

Nuclear export is also receptor-mediated and depends on a nuclear export signal, or NES.

1. A nuclear cargo protein containing an NES binds to an exportin receptor.
2. Exportin binds Ran-GTP in the nucleoplasm, forming a stable export complex containing cargo, exportin, and Ran-GTP.
3. This complex interacts with FG-repeat nucleoporins and moves through the nuclear pore complex into the cytoplasm.
4. In the cytoplasm, Ran-GTP is hydrolyzed to Ran-GDP with the help of Ran GTPase-activating proteins.
5. Hydrolysis causes the export complex to dissociate and releases the cargo into the cytoplasm.
6. Exportin and Ran-GDP return to the nucleus, where Ran-GDP is converted back to Ran-GTP by the nuclear guanine nucleotide exchange factor.

The Ran gradient ensures that cargo binds exportin mainly in the nucleus and is released mainly in the cytoplasm.

Nuclear localization signals and nuclear export signals are sequence motifs that direct proteins through the nuclear pore complex.

These signals may be located anywhere within a protein and can sometimes be masked or exposed by conformational changes, protein modification, or binding to another protein. This allows cells to regulate the subcellular location of nuclear proteins.

The Ran GTPase cycle provides the energy and spatial information required for directional transport through nuclear pore complexes.

• In the nucleus, Ran is predominantly in the Ran-GTP form because the nuclear guanine nucleotide exchange factor converts Ran-GDP into Ran-GTP.
• In the cytoplasm, Ran is predominantly in the Ran-GDP form because cytoplasmic activating proteins stimulate GTP hydrolysis.
• During nuclear import, Ran-GTP binds importin and releases the imported cargo in the nucleus.
• During nuclear export, Ran-GTP promotes formation of the exportin-cargo complex in the nucleus.
• After export, cytoplasmic GTP hydrolysis dissociates the export complex and releases the cargo.
• Ran-GDP is returned to the nucleus and converted to Ran-GTP.

The unequal distribution of Ran-GTP and Ran-GDP across the nuclear envelope creates a chemical gradient that determines the direction of cargo loading and unloading.

Cells regulate nuclear protein import so that transcription factors enter the nucleus only under appropriate conditions.

• A transcription factor may contain an NLS that is exposed in its active form.
• In the inactive state, the NLS can be masked by binding to an inhibitory protein or by a conformational change.
• Signaling pathways may activate protein kinases that phosphorylate the transcription factor or its inhibitor.
• Phosphorylation can expose the NLS, promote dissociation from the inhibitor, or create a binding site for an import receptor.
• The activated transcription factor is then recognized by importin and transported through the NPC.
• Once inside the nucleus, it binds regulatory DNA sequences and changes gene transcription.
• Dephosphorylation, degradation, or exposure of an NES can later promote nuclear export.

This mechanism links extracellular signals to changes in gene expression by controlling the intracellular location of regulatory proteins.

Nuclear protein export is regulated at several levels:

• Exposure of the NES: An export signal may be hidden in an inactive protein and exposed after phosphorylation, ligand binding, or conformational change.
• Exportin availability: The abundance and activity of export receptors can influence the rate of export.
• Cargo modification: Phosphorylation, acetylation, ubiquitination, or other modifications can alter cargo binding to exportins.
• Protein-protein interactions: Binding partners may retain a protein in the nucleus or expose its NES.
• Ran gradient: Efficient export requires Ran-GTP in the nucleus and GTP hydrolysis in the cytoplasm.
• Cargo retention: DNA, RNA, nuclear bodies, and chromatin can retain proteins in the nucleus even when an NES is present.
• Signal-dependent control: Cellular stress, hormonal signals, or developmental cues can activate export pathways.

These mechanisms ensure that proteins are exported only when their nuclear functions are completed or when cytoplasmic activity is required.

The nucleus is mechanically and functionally connected to the cytoskeleton through protein complexes in the nuclear envelope.

• The LINC complex, consisting of SUN proteins in the inner nuclear membrane and KASH proteins in the outer nuclear membrane, spans the perinuclear space.
• SUN proteins interact with nuclear lamins and nucleoskeletal components.
• KASH proteins interact with cytoskeletal elements such as actin filaments, microtubules, and intermediate filaments.
• This connection transmits mechanical forces from the cell surface and cytoplasm to the nucleus.
• It helps position and move the nucleus within the cell.
• It contributes to cell migration, tissue organization, and mechanosensing.
• Mechanical signals can influence nuclear shape, chromatin organization, and gene expression.

Thus, the nucleus is integrated into the overall cytoskeletal network rather than being an isolated organelle.

All three cytoskeletal systems contribute to cell division, but they perform different roles.

• Microtubules: They form the mitotic spindle, which attaches to chromosomes through kinetochores and separates duplicated chromosomes toward opposite poles.
• Actin filaments: They form the contractile ring during cytokinesis. Together with myosin, the ring constricts the cell membrane and produces two daughter cells.
• Intermediate filaments: Nuclear lamins are phosphorylated and reorganized during nuclear envelope breakdown. Later, they are dephosphorylated and reassemble around the daughter nuclei.
• Coordination: Microtubules position the spindle and determine the division axis, while actin and myosin complete physical separation of the cytoplasm.

The coordinated remodeling of these systems ensures accurate chromosome segregation, nuclear reformation, and cytokinesis.

Motor proteins convert chemical energy from ATP hydrolysis into directed movement along cytoskeletal filaments.

• Kinesins generally move cargo toward the plus ends of microtubules, often from the cell center toward the cell periphery.
• Dyneins generally move cargo toward the minus ends of microtubules, often toward the centrosome or cell center.
• Myosins move along actin filaments and transport vesicles, organelles, and protein complexes.
• Motor proteins contain a filament-binding motor domain and a cargo-binding region.
• They may transport membrane vesicles, mitochondria, chromosomes, mRNA-protein complexes, or entire organelles.
• Adaptor proteins connect motors to specific cargoes and regulate when transport occurs.

The polarity of microtubules and actin filaments provides directional information, allowing cells to organize long-distance and short-distance intracellular transport.