cell bio FINAL: nucleus part 2

describe the roles of the following components of the nucleus:

a) nuclear lamina

b) perinuclear space

c) nuclear pores

a) a meshwork of intermediate filaments beneath the inner nuclear membrane that provides structural support and organizes peripheral heterochromatin.

b) located between inner and outer nuclear membranes, serves as a buffer for protein and lipid exchange between the nucleus and ER

c) are multiprotein complexes embedded in the nuclear envelope that mediate selective bidirectional transport of RNAs, proteins, and ribonucleoprotein complexes between nucleus and cytoplasm.

what are lamins and why are they important? (list the types of lamins too)

they are filament proteins that form the nuclear lamina, providing structural support to the nucleus and helping organize chromatin

--------> Lamin A contributes to nuclear stiffness

--------> Lamin B is essential for nuclear assembly and anchoring the lamina to the inner nuclear membrane.

describe how integral membrane proteins of the nuclear membrane interact with both the nuclear lamina and chromatin

integral membrane proteins interact with the nuclear lamina through direct binding to lamins

--------> connect to chromatin by binding DNA or chromatin-associated proteins, anchoring chromatin at the nuclear periphery and maintaining nuclear architecture.

list two human diseases associated with nuclear envelope abnormalities and how the NE is affected in each scenario

Progeria:

- mutation in Lamin A produces progerin, which disrupts the nuclear lamina and causes misshapen nuclei, leading to premature aging.

Emery-Dreifuss Muscular Dystrophy (EDMD):

- mutations in Emerin (integral protein) or Lamin A/C weaken the nuclear envelope, altering nuclear structure and impairing muscle cell function.

list what nucleoporins are and their role in the nuclear envelope

- Nucleoporins are the individual protein components that make up nuclear pore complexes in the nuclear envelope.

- They form the structure of the pore and mediate selective transport of RNAs, proteins, and ribonucleoprotein complexes between the nucleus and cytoplasm.

list the three main types of nucleoporins and what their roles are

1) Scaffold nucleoporins:

- Form the structural framework of the nuclear pore complex, maintaining its shape and anchoring it within the nuclear envelope.

2) FG-repeat nucleoporins (channel nucleoporins):

- create a selective barrier, allowing transport receptors to shuttle cargo while blocking nonspecific molecules.

3) Transmembrane nucleoporins:

- span nuclear envelope and anchor the nuclear pore complex to the inner and outer nuclear membranes, stabilizing its position

a) what is meant by the "disordered region of the channel nucleoporins"?

'b) within the disordered region of channel nucleoporins, what is the role of the nuclear basket, the scaffold nucleoporins and the membrane ring proteins?

a) is the flexible, unstructured FG-repeat domains that form a selective, dynamic barrier for molecular transport through the nuclear pore.

b)

- Nuclear basket: Captures and organizes cargo exiting the nucleus, aiding in efficient transport.

- Scaffold nucleoporins: Maintain the structural framework of the pore, providing stability to the disordered channel.

- Membrane ring proteins: Anchor the nuclear pore complex to the nuclear envelope, keeping the channel correctly positioned.

what is the role of the alpha solenoid-beta propellor in Nups (nucleoporins) and how does it interact with them?

- provides a rigid structural framework within nucleoporins, helping maintain the overall architecture of the nuclear pore complex.

- interact with other Nup subunits to organize and stabilize the assembly of the nuclear pore, ensuring proper function and transport.

list some similarities between Nups and vesicle coats (like COPII, COPI, clathrin)

- Both use alpha-solenoid and beta-propeller domains to form rigid structural scaffolds.

- Both assemble into modular, symmetrical complexes that shape a central channel or vesicle while allowing selective transport or cargo capture.

- Both interact with membranes and cargo-binding proteins, anchoring the structure and facilitating the movement of molecules either through the nuclear pore or in vesicle trafficking.

describe how nucleoporins interact with the NPC (nuclear pore complex)

Nups form the structural and functional components of the NPC, creating selective channels and interacting with transport receptors to mediate cargo passage between the nucleus and cytoplasm.

describe what happens in each of the following scenarios:

a) localization of T-antigen containing its normal nuclear import signal

b) localization of T-antigen containing a mutated nuclear import signal

a) T-antigen is recognized by importins and efficiently transported into the nucleus, where it performs its normal nuclear functions.

b) altered amino acid sequence (Lys to Thr) prevents importin recognition, causing the T-antigen to remain in the cytoplasm and fail to enter the nucleus

there are four different cargo proteins that are involved in nuclear import. describe what they are attached to and where they are directed to

- cargoes 1-3 are attached to a nuclear import receptor (proteins that shuttle essential cargo from cytoplasm to nucleus) via a nuclear localization signal

- cargo 4 is attached to a nuclear import adaptor protein and a nuclear import receptor

why is it that cargo 4 is attached to a nuclear import adaptor protein AND a nuclear import receptor?

because the adaptor binds the specific cargo while the receptor mediates transport through the nuclear pore complex.

----------> cargos 1-3 have nuclear localization signals that are sufficient for direct receptor binding and import.

use the following terms in describing how cargo is BIDIRECTIONALLY imported / exported into the nuclear space:

- import receptors

- FG binding sites

- cargo

- FG repeats

- cytosolic fibrils

- Import receptors bind cargo in the cytoplasm and interact with FG repeats, which bind to FG binding sites on nucleoporins to move the cargo through the nuclear pore.

- import receptors navigate the pore channel, guided by cytosolic fibrils, while the repeated FG repeat-FG binding site interactions facilitate passage.

- After cargo delivery, the receptor returns to the cytoplasm, allowing bidirectional transport through repeated FG-mediated interactions.

describe how Ran-GEF and Rab-GAP interact with the Ran-GTP / Ran-GDP process

- Ran-GEF (in the nucleus) promotes the exchange of GDP for GTP on Ran, generating Ran-GTP that drives nuclear import and export directionality.

- Ran-GAP (in the cytoplasm) stimulates hydrolysis of GTP on Ran, converting Ran-GTP to Ran-GDP via GTPase and allowing import receptors to release cargo.

describe nuclear import now IN DETAIL including the RanGTP processes and cargo loading / unloading

1) a cargo protein with a nuclear localization signal (NLS) binds to the import receptor in the cytoplasm

2) the import receptor-cargo complex moves through the nuclear pore via interactions with FG repeats.

3) once inside the nucleus, Ran-GTP binds to the import receptor, causing the receptor to release the cargo.

4) the import receptor-Ran-GTP complex returns to the cytoplasm, where Ran-GAP stimulates GTP hydrolysis

----------> converts Ran-GTP to Ran-GDP & releases the receptor to start another round

describe how nuclear export occurs including the RanGTP processes and cargo loading / unloading

1) cargo protein with a nuclear export signal (NES) binds to an export receptor (exportin) together with Ran-GTP inside the nucleus.

2) the cargo-exportin-Ran-GTP complex moves through the nuclear pore via interactions with FG repeats

3) once in the cytoplasm, Ran-GAP stimulates GTP hydrolysis, converting Ran-GTP to Ran-GDP and causing the export receptor to release the cargo.

4) export receptor returns to the nucleus to start another round of export, while Ran-GDP is transported back into the nucleus for nucleotide exchange by Ran-GEF.

a) briefly describe how Ran is recycled

b) briefly describe how nuclear transport is regulated (import vs export)

a) via hydrolysis to Ran-GDP in the cytoplasm by Ran-GAP and then transported back into the nucleus for nucleotide exchange by Ran-GEF.

b) via the Ran-GTP/Ran-GDP gradient, ensuring that import and export receptors release or bind cargo in the correct compartment.

Describe how you might inhibit the nuclear import process.

HINT: think of something other than simply removing one of the essential proteins

1) mask the nuclear localization signal (NLS) on the cargo protein using a binding peptide or small molecule, preventing import receptors from recognizing and binding the cargo.

2) Disrupt the Ran-GTP gradient by inhibiting Ran-GEF in the nucleus or Ran-GAP in the cytoplasm, which would block proper directionality and cargo release.

If your evil goal was to make a pore that more or less looks like a pore, but has lost all selectivity for transport, what would you change?

1) Remove or mutate the FG-repeat nucleoporins, eliminating the selective FG-receptor interactions that normally filter what can pass through the pore.

2) Disrupt the scaffold structure that maintains the central channel, widening or destabilizing it so molecules diffuse freely without regulation.

explain how regulated nuclear import is a step in signal transduction via changes in calcium levels

- in an active T cell, high cytosolic calcium activates the protein phosphatase calcineurin

---------> dephosphorylates NF-AT (nuclear factor of activated T cells) and exposes its nuclear import signal.

- Dephosphorylated NF-AT is imported into the nucleus, where it activates transcription of target immune-response genes.

explain how regulated nuclear export is a step in signal transduction via changes in calcium levels

- as Ca2+ levels drop after transcription begins, calcineurin becomes inactive, allowing kinases to re-phosphorylate NF-AT and expose its nuclear export signal.

- re-phosphorylated NF-AT is exported back to the cytoplasm, completing a Ca2+-regulated cycle of nuclear import / export