Dr Shackleton's Work Flashcards Preview

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Flashcards in Dr Shackleton's Work Deck (40):
1

Why is the DNA contained in the nucleus?

Provides control of gene expression
Regulation of timing of DNA replication and mitosis
Organization of huge and complex eukaryotic genomes

2

What are the main features of the nucleus?

Nucleolus
Peripheral heterochromatin
DNA
NPC
Nuclear envelope
Nuclear lamina

3

What does peripheral heterochromatin do?

Act as a silencing environment

4

What is the nucleolus responsible for?

Transcription and processing of non-coding RNAs
Ribosome subunit assembly
DNA loops rich in rRNA genes forma structura component

5

Outline the basic NPC structure

Nuclear basket - regulate what passesthrough
8 fold symmetry
Multiple copies of 30 different nucleoporins
Aqueous pore in centre

6

How do small molecules (less than 40kDa)get in/out of the nucleus?

Diffuse through the NPC

7

How do large proteins get into the nucleus?

1. Cargo binds to importin in the cytoplasm
2. Goes through NPC
3. In nucleus Ran GTP binds displacing cargo
4. Goes through NPC
5. GAP activates Rans GTPase function causing Ran GDP to dissociate

8

What does the classical NLS constitute?

1/2 short stretches of basic amino acids (Arg, Lys, His) which binds to importin alpha

9

What is the most well known NES?

Leucine rich which binds to CRM1

10

Outline the process of nuclear export for a protein greater than 40kDa

1. Cargo and Ran GTP binds to the exportin in the nucleus
2. Ran GAP activates GTPase activity in Ran causing cargo and Ran GDP to dissociate
3. Exportin goes back through NPC

11

What are the methods for preventing nuclear export/import?

Masking the NLS/NES by:
Inhibitory Protein Binding
Post-translational modification
Phospohorylation
Tethering

12

Give an example of inhibitory protein binding to prevent nuclear import

IkB binding to NFkB

13

Give an example of how phosphorylation can prevent nuclear export

Cyclin B1 - phosphorylation prevents it being exported stimulating onset of mitosis

14

Outline the structure of microtubules

Alpha/beta tubulin dimers polymerise to form a protofilament, 13 of which form a microtubule

15

Outline microtubule treadmilling

Dimers are added at the positive end and removed at the negative end

16

In vivo what stabilises microtubule treadmilling normally?

GTP cap on the positive end
Gamma tubulin ring on the negative end

17

Outline catastrophe and rescue

Microtubules depolymerize 100x faster from an end with GDP-tubulin therefore if the GTP cap is lost at the positive end causes rapid depolymerisation. Eventually the cap is restored

18

Name two drugs that act as MAPs

Taxal and Colchicine

19

What are the 6 actin isoforms of mammals?

4 alpha - contractile structures
Beta - cell cortex and lamellipodium
Gamma - Stress fibres

20

Outline the structure of actin microfilaments

G-actin polymerises to form f actin, which consists of two parallel helical structures

21

Outline actin microfilament treadmilling

Allows rapid reorganization, normally between C+c and C-c therefore leads to treadmilling
Addition at positive end = loss at negative end

22

What does tropomodulin do?

Block the negative end of actin microfilaments

23

What toxins inhibit actin polymerisation?

Cytochalasin D - blocks positive end
Latruncilin - Prevents G-actin formation

24

How is actin nucleation controlled?

Formins nucleate long unbranched actin filaments. When activated by Rho-GTP the formin straightens out allowing G-actin to access it's FH1 domain

25

What does fimbrin do?

Moulds actin into microvilli, filopodia and focal adhesions

26

What does alpha actinin do?

moulds actin into Stress fibres, filopodia and muscle Z line

27

What does spectrin do?

Support the cell membrane

28

What does filamin do?

Moulds actin at the leading edge, stress fibres and filopodia

29

What does ezrin do?

Attach microvilli actin to the plasma membrane

30

What are the steps of cell migration?

Extension -> Adhesion -> Translocation ->De-adhesion and endo-cytotic pathway

31

How do proteins establish polarity in migration??

Rho is only active at the rear end
Cdc42 establishes polarity

32

Give the structure of intermediate filaments

Curved with an alpha helical doain
Dimerize via the alpha helix, 2 dimers form anti-parallel tetramers = Protfilaments, 4 protofilaments = 1 protofibril

33

What are the main characteristics of intermediate filaments?

Non-polar
More stable than actin microfilaments/microtubules
Very elastic
Bend easily
Provide tensile strength and resistance to mechanical stress

34

What genes are lamins encoded by?

LMNA - A
LMNB1- B1
LMNB2 -B2

35

What happens to nuclear lamins upon entering mitosis?

Become hyperpolarised causing disassembly

36

What do nuclear lamins do?

Provide structural support and organize chromatin

37

What are laminopathies?

Mutations in LMNA
e.g. Emery-Dreifuss muscular dystrophy -> makes nuclei susceptible to rupture
e.g. Hutchinson Guilford progeria syndrome -> toxic effects of farnesylated lamin A, lamina loses elasticity and early senscence

38

What is the structure of keratin?

Heterodimers of acidic and basic/neutral chains with disulphide bonds providing strength

39

What are desomosomes?

Structures made of keratin which connect adjoining cells in the epithelia

40

What do mutations in keratin lead to?

KRT5 and KRT14 -> Epidermolysis bullosa simplex