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Cytosol Nucleus Tracking Flashcards

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1
Q

Slide 4

A

Scanning EM image of nucleus nuclear envelope, cytoplasm

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2
Q

What is the perinuclear space with?

A

Endoplasmic Reticulum

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3
Q

How many nuclear membranes are there?

A

“2” - outer & inner!

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4
Q

What else is in the nuclear envelope?

A

Pore complexes!

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5
Q

Fibrous Lamina (or Nuclear Lamina)

A

A meshwork of intermediate filament proteins lining the inner aspect of the inner nuclear membrane

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6
Q

Which materials are exchanged between the nuclear and cytoplasmic compartments?

A 
Slide 6
Nuclear proteins (in)
Ribosomal proteins (in)
Ribosomal subunits (out)
mRNA (out)
tRNA (out)
Shuttling proteins (out)
Ions (both)
ATP (both)
Smallecules (
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7
Q

Nuclear Envelope Components (Start presentation here?)

A

Nuclear Membranes
Nuclear Lamina
Nuclear Pore Complex
Slide 10 is helpful

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8
Q

Outer Nuclear Membrane

A

Continuation of RER, has ribosomes on its outer surface

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9
Q

Inner Nuclear Membrane

A

Associated with Nuclear Lamina & Chromatin

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10
Q

Pore Membranes

A

Connect the two!

Associated with pore complexes!

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11
Q

Nuclear Lamina

A

Slide 11 & 12

Made of 10 nm-diameter fibers (characteristic of IFs)

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12
Q

Components of a nuclear lamin IF

A

Variable Head & Tail Domains

Highly Conserved alpha-helical rod domains (responsible for dimerization), then higher polymerization into IFs

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13
Q

What makes Lamin IFs different from cytoplasmic IFs

A

They have more repeats in the rod domains

They have NLS

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14
Q

What happens to the nuclear lamina during mitosis?

A

The lamina proteins get phosphorylated, causing them to disassemble into free-floating dimers.

They later re-assemble by being dephosphorylated.

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15
Q

What proteins does the lamina interact with in the inner nuclear membrane?

A 
LBR
Emerin
POM121
gp210
Myne-1
Nurim
Nuclear Pore Complex
MAN1
LAPs
HETEROCHROMATIN
60 - 80 proteins, total
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16
Q

What are the two types of lamins that make up the Nuclear Lamina?

A

A-Type

B-Type

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17
Q

Nuclear Pore Complex

A

Slide 15

Slide 16 - Pretty!!!! (dissolved membrane away with detergent, but the pore complexes remain)

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18
Q

Nuclear Pore Complex Components

A 
Slide 17!!!!
Cytoplasmic Fibrils
Cytoplasmic Ring
Nuclear Ring
Nuclear Basket
Distal Ring
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19
Q

What proteins make up a Nuclear Pore Complex?

A

Nucleoporins (many have FG repeats)
Integral Membrane Proteins (gp210, POM121)
Associated Proteins (Ran, Lamins)
Others not well characterized

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20
Q

Mechanisms of Nuclear Transport

A

Diffusion

Active Nuclear Transport

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21
Q

Diffusion

A

Chemical and electrical gradient equilibrium
Works in NPC for smallecules like ions and ATP
Limited by permeability of membrane and diameter of pores

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22
Q

Rate of Diffusion

Study These Flashcards
A 
Inversely proportional to square root of the molecular mass.
Some molecules (like histones) can diffuse, but not as quickly as you'd like, so there are alternative mechanisms.
23
Q

Diffusion Size Cutoffs

Study These Flashcards
A

60 kDa and above can’t diffuse into nucleus

24
Q

Some molecules concentrate in the nucleus, even though they could freely diffuse. Why?!

Study These Flashcards
A

They bind to thangs in the nucleus. Ex: Cations that bind to negatively-charged DNA

25
Active Nuclear Transport
Faster uptake for larger molecules that could diffuse, but slowly. Uptake of molecules to large to diffuse Requires ATP or GTP
26
NLS code
PKKKRKV Pro-Lys-Lys-Lys-Arg-Lys-Val KKKRK is the most important Mess up something in there, and the NLS won't werq Discovered from the SV40 Large T Antigen (viral) Also, hella basic
27
What happens when you covalently attach to albumin or gold?
GOLD ASS NUCLEUS
28
A few examples of NLS sequences
``` PKKKRKV GKKRSKA KAKRQR RKRRR PRRRK PYLNKRKGKP KKKKRKREK KKKRRSREK ```
29
Second type of NLS
Bipartite (discovered in Nucleoplasm) KR PAATKKAGQA KKKK Two basic clusters separated by a sequence of about 10 rando amino acids
30
Two step process of nuclear import
Step 1 - Energy Independent | Step 2 - ATP Dependent
31
Step 1
Fraction A Binding of NLS-containing substrate to to a nuclear pore complex NEM-sensitive
32
Step 2
Fraction B Translocation - Moving of the substrate across the pore. Blocked by non-hydrolyzable GTP analogues
33
Fraction B
Contains Ran (Ras-like protein that binds GTP) Restored import in the presence of ATP and Fraction A. Recombinant Ran also active. Blocked by non-hydrolyzable GTP analogues
34
What does this mean?
It was primarily Ran-dependent hydrolysis of GTP that provides energy for transport across the pores
35
Fraction A
``` Heterodimeric complex needed for NLS-mediated import Karyopherin alpha/Karyopherin beta Importin alpha/importin beta NLS receptor/p97 NPC targeting complex ``` THESE ARE ALL NAMES FOR THE SAME COMPLEX Typically karyopherin or importin are the running names these days.
36
Karyopherin/Importin Alpha - Important structure
N-terminal, C-terminal, IT IS A PROTEIN Functional shit: NLS Binding/Contact Surface Karyopherin/Importin Beta1 binding domain
37
Karyopherin/Importin Beta1 - Important structure
The first of a family of karyopherinBetas/ImportinBetas Involved in SV40 Large T antigen and Bipartite NLS 90 kDa Binds importin-alpha (via importin beta binding domain) Binds RAN-GTP Binds FG repeats in nucleoporins There is a large family of these proteins. Some mediate import. Some mediate export. Beta1 needs Alpha (middleman) to bind to the NLS. Most other karyopherin Betas bind directly to cargo. They bind to: Ribosomal proteins tRNA Some are involved in export, but rely on an NES for that.
38
NES
Nuclear Export Sequence Very Leucine-rich. The tRNA one appears on the tRNA itself, not a bound protein
39
"Important things" about Karyopherin/importin(exportin)-Betas
Over 20 in the human genome Each receptor recognizes a different type of targeting signal for import or export Soluble Interact with both Cargo (sometimes indirectly) and NPC Shuttle back and forth through NPC carrying cargo one way, then recycling back without cargo (usually)
40
mRNA export
Not completely understood. Coupled to splicing somehow. Inhibit splicing, inhibit export. No karyopherin/importin involved.
41
Ran-GAP
Activates the cytoplasmic GTPase so Ran-GDP predominates in cytoplasm
42
Ran-GEF
Catalyzes exchange of GDP for GTP.
43
Importins release their cargo when?
Upon binding Ran-GTP
44
Exportins bind their cargo when?
When they bind to Ran-GTP
45
Exportins release their cargo when?
When Ran-GTP is no longer around.
46
NLS and NES signals can be masked.
Phosphorylation - conformational change | Phosphorylation - binding to another protein that hides the signal
47
NLS and NES signals can be created
Phosphorylation - Conformational change Phosphorylation - Release fo masking protein Degradation of bound masking protein
48
What does the nuclear envelope do besides all this?
Regulates transcription factors through MAN1 and LEMD3
49
MAN1/LEMD3
Brake Resides in inner nuclear membrane Downregulates Smad 1/2/3
50
Smads 1/2/3
Move into the nucleus (using karyopherins) in response to activation of the TGF-Beta signaling family
51
What happens if you lose half your MAN1?
Sclerosing Bone Dysplasias with Skin Abnormalities. Buschke-Ollendorff Syndrome and Osteopoikilosis From increased TGF-Beta and BMP signaling in bone and skin
52
Osteopoikilosis and Buschke-Ollendorff
Hyperdensities in cortical bone (can be painful) From overactive BMP and TGF-Beta laying down the cortical bone. Also has skin effects too.
53
Last slide
Um I don't think it's on the test. memorize it maybe? I think maybe it was just to make this all seem relevant.

Biomolecules & Cells (2 decks)

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