Lecture 2 - Nuclear Transport and Structure

Nucleolus

 a non-membrane bound compartment within the nucleus organized around Nucleolar Organizing Regions (NORs) on acrocentric chromosomes, formed as a consequence of rRNA synthesis and ribosome assembly

Three zones of the Nucleolus

Fibrillar center, Dense Fibrillar Component, and Granular Component

Fibrillar Center

(rDNA transcription by RNA polymerase I)

Dense Fibrillar Component

(rRNA splicing and processing)

Granular Component

(pre-ribosomal subunit assembly with ribosomal proteins) 

Nuclear Lamina

A fibrillar protein meshwork located just underneath the inner nuclear membrane, composed of intermediate filament proteins known as lamins. 

Lamins

form long, coiled-coil rod-like dimers/tetramers that provide tensile strength and structural integrity to the nuclear envelope

Nuclear Pore Complex (NPC)

A 120 MDa macromolecular assembly displaying 8-fold symmetry that spans the double lipid bilayer of the nuclear envelope

It is constructed from about 30 distinct nucleoporins (Nups), providing an asymmetric structure with cytosolic filaments on the cytoplasmic side, a central core scaffold, and a nuclear basket extending into the nucleoplasm. 

Describe the organizing principles for chromosomes in the eukaryotic nucleus.

Chromosomes occupy distinct, non-random regions in the interphase nucleus known as chromosome territories (CTs). 

NOR-containing regions group at the nucleus, and telomeres associate with the nuclear lamina at the periphery. 

Relationship between chromosomal organization or location within the nucleus and gene activity

The nuclear is spatially compartmentalized regarding expression. 

• Gene-poor, condensed heterochromatin enriched in repressive marks (H3K9 methylation) is sequestered at the nuclear periphery near the lamina. 

• In contrast, gene-rich, actively transcribed euchromatic regions populate the nuclear interior

Gene gating hypothesis

Hypothesis postulated that transcribable (expanded) regions of the genome interact directly with specific nuclear pore complexes on the nuclear periphery, so that all transcripts from a given gated gene would exit through that dedicated pore.

Only partially true; actively transcribed genes do alter their spatial positioning, but they move into the nuclear interior rather than tethering directly to the nuclear pore.

How is GFP technology used to track the behavior of genes in response to transcriptional activation?

1. An inducible locus-tracking system utilizes a lac operator (lacO) array inserted into a heterochromatic genomic site

2. Cells express an eGFP–lac repressor–FKBP3​ fusion protein (which fluorescently marks the lacO array) and an FRB-VP16 transcriptional activation domain fusion

3. Addition of rapamycin chemically reconstitutes the functional transcription factor by heterodimerizing FKBP and FRB.

4. GFP fluorescence microscopy then enables real-time tracking, demonstrating that transcriptional activation induces long-range directional movement of the locus from the silent nuclear periphery into the nuclear interior within ~2 hours

What is the primary function of lamins?

Lamins maintain nuclear structural integrity, anchor nuclear pores, provide mechanical tensile strength, organize interphase chromosomes, tether telomeres/heterochromatin to the envelope, and participate in gene regulation and cell cycle control.

What molecule is defective in individuals with Progeria?

Caused by a dominant mutation in the LMNA gene (Lamin A/C) producing a truncated, toxic form of Lamin A protein (progerin). 

Why does mislocalization of specific chromosomal structures lead to Progeria?

Toxic Lamin A disrupts the attachment of telomeres to the nuclear lamina at the periphery. 

Mislocalization of telomeres impairs their proper replication, accelerating telomere shortening and driving cells into premature cellular senescence and cell

Describe the structure of the nuclear pore

Composed of cytosolic filaments, outer/inner/luminal core rings, a central channel (~10 nm unstretched), and a nuclear basket

What is the relevance of Nucleoporins?

Proteins rich in unstructured Phenylalanine-Glycine (FG) repeats that line the central channel. 

They form a floppy, flexible diffusion barrier ("sea of FG repeats") that blocks non-specific transport while serving as transient docking/binding sites for transport receptors (β-karyopherins).

Passive Diffusion

Small molecules and proteins <5 kDa freely and rapidly equilibrate through the NPC. Molecules up to 40–60 kDa can diffuse passively, albeit progressively slower.

Gated/Active Transport

Cargos >40–60 kDa (and up to >30 nm in size) are gated out and cannot pass via passive diffusion; they require a Nuclear Localization Signal (NLS), a soluble transport receptor (β-karyopherin), and energy input via GTP hydrolysis

GTPase cycle

GTPase switches between two states:

GTP-bound = ON/ACTIVE
GDP-bound = OFF/INACTIVE 

1. GDP-bound GTPase → inactive

2. GDP is exchanged for GTP → active

3. GTP is hydrolyzed → GDP

4. GTPase returns to inactive state

The effect of accessory factors

GAPs, GEFs, and GDIs

GAPs (GTPase Activating Proteins)

Stimulate intrinsic GTPase activity to hydrolyze GTP → GDP + Pi​, turning the switch OFF

GEFs (Guanine Nucleotide Exchange Factors)

Catalyze the dissociation of GDP to allow GTP binding, turning the switch ON.

GDIs (GDP Dissociation Inhibitors):

Inhibit the release of bound GDP, maintaining the switch in the OFF state.

Nuclear import cycle (NLS, carriers, release)

1. A cytosolic β-karyopherin (importin) binds the NLS of a cargo protein.

2. The receptor-cargo complex transits through the NPC by making transient, diffusive interactions with FG-Nups.

3. In the nucleus, RanGTP binds directly to the β-karyopherin, inducing a conformational change that triggers the release of the NLS cargo.

4. The RanGTP–karyopherin complex exports back to the cytoplasm, where RanGAP stimulates GTP hydrolysis, causing RanGDP to dissociate and freeing the receptor for another cycle.

Role of Ran asymmetry in concerted transport

RanGEF (RCC1) is bound to nuclear chromatin (producing high nuclear RanGTP)  while RanGAP is tethered to the cytosolic face of the nuclear envelope (producing high cytosolic RanGDP).

This steep RanGTP/RanGDP gradient across the nuclear envelope provides directional driving force for cargo loading and unloading.

What type of assays were developed to elucidate the process?

Elucidating assays: Uses digitionin-permeabilized cells

• Digitonin selectively perforates the plasma membrane while leaving the nuclear envelope intact, stripping out cytosolic components. 

• Reconstituting import required adding back fluorescent NLS-cargo, GTP, and fractionated cytosolic extract (using ion-exchange, gel-filtration, and affinity chromatography) to isolate required soluble factors like Ran and karyopherins