Exam 4 - Dave - Nuclear Structure, Function, and Transport

One nuclear pore has about how many proteins

1200

With Cryo-EM, one can see what rings

cytoplsmic, luminal, inner, and nuclear

The outer nuclear membrane is continuous with

the rough ER

ONM proteins have high concentration and bind

cytoskeleton

The inner nuclear membrane proteins bind

nuclear lamina

INM is joined to ONM at the

Nuclear Pore Complex

Perinuclear Cisternae is the region between

outer/inner nuclear membrane and contagious with ER lumen

What is unique about nuclear pores

they allow molecules to go both ways without folding and unfolding

How does the nuclear pore allow molecules to go both directions

because of the specific NLS and NES present

Tripartite Nuclear Localization Signals

EGFRs and HER2s

What is a hallmark of a eukaryotic cell

separation of the genome from the sites of mRNA translationand protein synthesis by the nuclear envelope.

The ONM is functionally similar to

ER membranes

The ONM has what bound to its cytoplasmic surface

ribosomes

The INM is lined by the

nuclear lamina

The INM serves as an ____ for chromatin

attachment site

The inner nuclear membrane will bind what two components

nuclear lamina and chromatin to maintain nuclear structure.

The nucleoplasm is enclosed by the

nuclear envelope and contains chromatin and nucleolus.

The INM and ONM are continuous at the

nuclear pores that regulate transport between the nucleus and cytoplasm.

The INM and ONM are continuous at nuclear pores, which allows for

the flux of lipids and proteins during nuclear expansion

Which microscopy is best to see the nuclear pore complex

STMand EM provide high-resolution images.

Nuclear pore complex has what symmetry

8-fold radial symmetry

The Nuclear pore Complex is ringed by

protein particles

NPC proteins are termed

Nups (nucleporins)

The NPC has what directional form of transport

bidirectional

NPCs are the sole channels for

small polar molecules, ions, proteins, and RNA

Within the NPC, polar molecules at what size are freely diffusable

≤ 5 kDa and slows till 60kDa max

NPC-nucleoporin has how many structures

6

Transmembrane Ring proteins

anchors Nuclear Pore Complexs to Nuclear Envelope

Scaffold nucleoporins (layered rings)

bend membrane and insert NPC

Channel nucleoporins

line central pore with tangled channel IDRs

Channel IDRs mechanism

F/G repeats → meshwork → blocks molecules 60kDa ≤

IDR fibrils will form the

nuclear basket

IDR fibrils mechanism

Nuclear fibrils converge → nuclear basket/fish-trap → fibrils open → optimal transport

Cytosolic fibrils

trap and increase mass action for nuclear transport

Macromolecular transport across NPCs occurs through an ____ and NOT directly through double-layered membrane

aqueous pore

NPCs can transport fully folded assembled proteins in/out of the nucleus as

a particle

How to visualize active import at NPCs

Colloidal gold particles (GPs) coated with NLS-peptides enter the nucleus

Because collonidal gold particles have large diameters so they are imported into the NPC by

active transport

EGFR Tripartite-NLS promote nuclear import of

Pyruvate Kinase (which is a cytoplasmic protein)

In resting T-cells, phospho-NFAT (P-NFAT) is

cytosolic

T-cell activation raises

cytosolic Ca2+ levels

In high Ca2+ levels, Calcineurin will dephosphorylate and bind NFAT, which will

mask NES, expose NLS and activate genes

In low Ca2+ levels, the NFAT/Calcinenurin disassociates and

NFAT dephosphorylates, inactivating NLS and NFAT to relocate to the cytosol

In high cholesterol, the SREBP/SCAP complex anchors to the

ER membrane cholesterol via SCAP

RNA export route: tRNA

Exportin-t/Ran-GTP

Nuclear export: miRNA

Exportin 5/Ran-GTP

Nuclear export: snRNA

CBC/ALY/NXF1

Nuclear export: rRNA

CRM1/Nmd3/Ran-GTP

NES-containing adaptors

PHAX (snRNA), ALY/NXF1 (mRNA), and Nmd3 (rRNA)

Exportins, but NOT importins, are needed for RNA because

RNA only comes out of the nucleus

NES signals

PHAX, ALY, NXF1, Nmd3, and CBC

SUN protiens are present in the

inner nuclear membrane

KASH proteins are at the

outer nuclear membrane

SUN-KASH domains bind within the

perinuclear space

SUN proteins connect the

nuclear lamina/chromosomes

KASH proteins connect to the

cytoskeleton at the microtubules or actin filaments

SUN-KASH functions (6)

Mechanically couple the nucleus to the cytoskeleton, Involved in chromosome movements, Regulates meiosis, Nuclear and centrosome positioning, Nuclear migration, and Global cytoskeletal organization

Nuclear lamins are made up of

type V intermediate filaments

The Nuclear Lamina is located at

the nuclear side of the inner nuclear membrane

The nuclear lamina functions

support, stability, anchoring for complex formation

Nuclear lamina can provide structural and functional link between DNA and nuclear envelope by directly interacting with

chromatin

Lamins are unique to

Metazoans

A-type lamins

Lamin A and C proteins isoforms in terminally diff. cells

B-type lamins

Lamin B1 and B2 in all cells generated by two genes (B1 and B2)

Lamin C does NOT have a

CAAX-box

Lamin dimers are formed by

monomers associating in a parallel, head-to-head, fomring a coiled-coil central a-helical domain

Lamin polymers assemble by

dimers assembling in a polar head-to tail manner wiht ~4nm overlap

Protofilaments of lamins form by

anti-parallel assembly of two lamin polymers to have 4 protein per unit

Intermediate filament of lamins is formed by

four protofilaments assemble laterally to have about 16 proteins per unit

At the inner nuclear membrane, high order lamins anchor via

prenylation of its C-terminal cystein and binding to proteins Emerin and Lamin B-receptor

In the nucleoplasm, higher order lamin polymers anchor to the

chromatin

Lamins are prenylated at the

C-terminal Cystein residue

Where is the Progeria lamin mutation

in exon 11 due to incorrect splicing/truncation

What causes Progeria

protease cleavage defect in LaminA that leads to permanently prenylated progerin

constitutive LADs (cLAD) are gene-poor and remain associated with

the lamina in all cells all the time

Facultative LADs (fLADs) become attached and move to the center of the

nucleoplsm during developmental cell specification

Nucleolus-associated chromatin domains (NADs) are

gene-poor regions enriched for pericentric satellite A/T-rich repeats

What technology can be used to detect where the chroamtin protein binds

DamID utilizing unique m6A tag

During mitosis, the nuclear envelope

disassembles

In interphase, the breakdown and reformation of the NE/lamina is by

phos/dephosphoylation cycles of lamins, NPCs and INM proteins

In telophase, breakdown and reformation of the nuclear envelope happens by

Nuclear envelope forms adjacent to decondensing daughter chromosomes, eventually fusing to complete

Unlike metazoans, in yeasts the nuclear envelope remains

intact during mitosis

Interphase chromosomes are

diffuse chromosomes

Anaphase chromosomes are

condensed chromosomes

Chromosomes are topologically separated in

loops

Interphase chromosomes have

extended loops with active transcription

Transcription factories are clustered sites of

RNA polymerases and transcription factors where active chromatin are transcribed

Chromosomal territories are

distinct nuclear spaces

CT-A is what territory

Active or Euchromatin territory

CT-B is what territory

Repressed or Heterochromatin territory

Active TADs reside in the

nuclear interior and bind activators

Repressed TADs bind

repressors, have heterochromatin and attached to nuclear lamina

cLADs and fLADs are subset of

TADs

CTCF-Cohesion complex loops

chromatin and recuits enhancers adjacent to target-genes or insulate enhancers

The Nucleolus is a

membraneless organelle

Nucleolus are a site of

rRNA transcription, processing, and pre-ribosome assembly

The Nucleolus is NOT surrounded by

a membrane

TADs and 3-C technology is used to define

transcriptionally active chromatin loop interactions in 3D chromatin