Nuclear lamins, pores and biocondensates 1 Flashcards
1
Q
What fondue
A
Principles of phase separation
2
Q
Describe mammalian cell = gen
A
15mirons
Length and width
Many organelles
3
Q
Describe pm of mammalian cell
A
Some extensions at top of cell
4
Q
What is biggest organelle
A
Nucleus= size of rbc
Different from other organisms
5
Q
Define organelle
A
Section of cell that performs functions delineated by membranes
6
Q
What do we see as come from cytoplasm - organization of nucleus
A
Nuclear envelope
Nuclear lamina
Nuclear interior
7
Q
Describe nuclear envelope
A
Inner anf outer ncuelar membrane, perinuclear space
Nuclear pore complexes
8
Q
Describe inner and outer nuclear membranes - organization of nucleus
A
Outer
Perinuclear space
Inner
(2 lipid bilayers, membranes, differs in proteins they carry )
9
Q
Describe npcs - organization of nucleus
A
At junction of inner and outer nuclear membranes
Phase separation controls transport - some components of npc involved in phase sep
Important for trafficking most materials across membrane
10
Q
Describe nuclear lamina - organization of nucleus
A
Underlies inner ncuelar membrane
Composed of filaments
11
Q
Name parts of nuclear interior - organization of nucleus
A
Nucleolus
Chromatin
Nuclear bodies
Nuclear matrix
12
Q
Describe nucleolus - organization of nucleus
A
Formed by phase sep
Compartments
13
Q
Describe chromatin - organization of nucleus
A
Phase sep regulates transcription - proteins associated with chromtain undergo phase sep = controls transcription or splicing
14
Q
Describe nuclear bodies - organization of nucleus
A
Formed by phase separation - in nucleoplasm
No membrane, involved in splicing, dna repair and other functions
Many compartments
15
Q
Describe nuclear matrix - organization of nucleus
A
Filament system - inside nucleus, role in immobilizing processes = transcription splicing
If treat cells with salt/detergent,dnas, rnas = left with nuclear Matrix
16
Q
Describe spaces of cell - what is continuous with what
A
Outer ncuelar membrane - continuous with er membrane - no physical barrier to prevent molecules from travelling between spaces
Perinuclear space er lumen
17
Q
Describe nuclei and nucleolus
A
Nucleolus = inside nuclear lumen
2-3 nucleoli in mammalian cell
Not surrounded by membrane
Heterochromatin in nucleoplasm
18
Q
Describe antibody staining nucleoli
A
Nucleoplasm - no nucleoli
But own area
19
Q
Name all functions of nucleoli = 9
A
Produce and process 47s ribosomal rna,48s,45s = same
Assemble ribsomal subunits - nucleoli make the small and large ones
Make/assemble signal recognition particle =srp
Regulates apoptosis - control cell death
Control cell cycle progression
Regulate p53 function (tumour suppressor protein)
Control telomerase function
Regulate stress response
Play a role for replication of many viruses - health
20
Q
Describe what nucleolus divided into
A
Diff subcompartemnts
Formed by phase separation
21
Q
Name components of nucleolus
A
Granular component
Dense fibrillar component
Fibrillar component
22
Q
Describe granular component nucleolus
A
General background
Dense fibrillar compartment in it
23
Q
Describe dense fibrillar component nucleolus
A
Embedded in granular component
Has fibrillar component in it
24
Q
Describe fibrillar component nucleolus
A
Inside of dense fibrillar component
Has rna pol1 = polymerase, generated 47s precursor of ribsomal rna
25
Describe what rna pol 1 does
Rna pol 1
Transcribe 47s at border of fibrillar and dense fibrillar components
Then as primary transcript moves through nucleolus proteins added
At end = en up with ribosomal subunits - mature kinda, outside nucleolus
26
Describe properties of nucleoli
Drop like properties
Liquid liquid phase separation
Compartments = developed with drop like properties
Drops that can fuse or divide
27
Describe properties of nucleoli = exp and conclusions
Fbl, fibrillarin in dense fibrillar compartments
Npm, nucleophosmin, b23 in granular compartment
Mix fbl and npm = generates droplets, not uniform, diff compartments which contain fbl or npm
= tendency to make liquid liquid droplets, also to separate from each other
= NUCLEOLUS FORMED BY PHASE SEP
28
Describe ribosomal biogenesis
COMBINING rRNA with ribosomal proteins
29
Describe large subunit rna
rRNA site of transcription
28s in nucleolus, 5.8s too
5s=outside nucleolus, in nucleus, made separately
Proteins all synthesized in cytoplasm=~47
30
Describe small subunit rna
18s made in nucleolus
~32 proteins in cytoplasm
31
What do human cells have - ribosome biogenesis
In each haploid = 5 clustered ribosomal rna genes
Cells have 10 nuclear organizers - NOR per diploid genome, (cluster dna active = forms nucleoli)
32
Describe rDNA
Organized into 43kb tandem repeats
33
What do human diploid cells have
~400 copies of rRNA
= encode 47s precursor
40% of genes active = generate de novo ribosomal subunits, accumulate 47s ribosomal rna = attract large variety processsing factors = help make ribosomal subunit
34
How are ribsomal subunits assembled
Vectorial fashion
35
Describe whole process of making ribosomes
Lop of nucleolar organizer dna - contains clusters of ribosomal rna
45s rRNA precursor - process rna and make 28s, 5,8s and 18s ribosomal rna
Modify rnas=add proteins from cytoplasm
Export out - npc - transport and activation creates functional ribosomes
MEANWHILE = 5s rRNA made outside nucleolus - in nucleoplasm, moves into nucleolus then added to other subunits
END UP WITH SMALL OR ALRGE SUBUNIT = THEN HAVE TO TRANSPORT FROM NUCLEOPLASM —> CYTOPLASM BY NUCLEAR PORE COMPELXES AND TFS
36
Why don’t we translate in nucleus
IF TOO big = cannot get through npc
Also if translate unsolicited rna = bad, gibberish, need spliced dna modified = processed transcript
37
Describe nucleoli and cancer
Linked to many diseases
High proliferation - have larger nucleus bc need lots of proteins so many ribosomes bc rapid cancer cell divisions, making proteins = need lots of stuff to divide
38
Do all proliferating cancer cells have enlarged nuclei
MANY BUT not ALL
Correlates with a higher rate of ribosome production
39
Describe cancer cells - gen
Proliferating cancer cells often more sensitive to inhibitors of rRNA synthesis than normal counterparts
40
What makes killing cancer cells with drugs that target nucleolus more effective
Higher the rate of ribosome production
41
Where is npc
Embedded in ncuelar envelope = between inner and outer membranes
42
Human npc size
120MD (size of 30 ribosomes= huge)
(Yeast npc = 60MD)
43
How does npc work
Looks diff on cytoplasm and ncuelar side
Important for trafficking/transport
Involved in active transport and diffusion in/out nucleus
Depends on side molecule
TRANSPORYT OF MOST BUT NOT ALL MATERAL ACROSS NCULEUS
44
Describe symmetry of npc
If slice perpendicular to membrane - 8 fold symmetry = identical pieces
45
Describe nuclear lamina of npc
Under inner nuclear membrane
46
Describe central gated Channel npc
Mediates protein trafficking + rna trafficking and diffusion
Has to be small molecules - <4kda = diffusion
If bigger = active transport
If super huge. = cannot use nuclear pore = use other methods
47
Describe how many proteins make up npc
At least 8 copies each protein
8 slices —> 35 diff proteins, many copies = add up all proteins = around 1000 proteins that make up npcs
48
Name the flavours of npc proteins
Np62
Proteins with fxf, glfg, no repeats
49
Describe fxf npc
Phenylalanine, any aa, phenylalanine
Function = protein transport in and out nucleus
50
Describe glfg repeat
Glycine, leucine, phenylalanine, glycine
Function = rna transport - mRNA export
51
Describe no repeat npc
Anchor npc to memrvane
52
Describe nuclear envelop and lamina
Under inner membrane
Filaments - nuclear lamins = intermediate filament proteins
Mass = 65-70kda
53
Are lamins only in nuclear lamina
Also lamins inside cell,= implicated in diseases
NOOOOOO
Inside cell
54
Describe nuclear lamina in detail
Contacts inner ncuelar membrane and chromatin in cells
- by set of proteins only near inner nuclear membrane
55
What are nuclear envelop proteins
Diff proteins that sit in inner ncuelar memrvane
Contact lamins and chromatin
56
Name the 2 types pf nuclear envelop proteins
LBR
LAP2
57
Describe LBR = nuclear envelop proteins
Multiple transmembrane regions
58
Describe LAP2 = nuclear envelop proteins
One tm domain span
59
Why are nuclear evenlop proteins important
Proteins specific to inner membrane
Provide network fo intelligence = includes lamin and chromatin
Important to organize chromatin, pull to nuclear envelop
Helps lamins and chromatin contact = helps control gene expression
60
Describe connection between nuclear lamina and actin cytoskeleton
Actin cytoskeleton + tubulin
Compartments need to talk ti each other
61
What are sun and Kash
Communicates between nculeuar interiors and cytoplasm
62
Describe sun
In inner nucelar membrane
Can also interact with proteins in nucleoplasm
Large domains in perinuclear space = interact wirh domains of kash proteins
63
Describe kash
Sit in outer nuclear memrvane
Large domains in cytoplasm
Can interact with proteins in cytoplasm
Interact with sun domains in perinuclear space/lumen
64
Describe linc
Contacts between inner ncuelar memrvane proteins and outer mem proteins = LINC = LINKER OF NUCLEOSKELETON AND CYTOSKELETON
65
What does kash interact with
SUN
ALSO WITH ACTIN AND MICROTUBULES = MOTORS IN CYTOPLASM
Links nucleoplasm and inner and outer ncuelar memrvnaes and cytoplasmic filaments
66
Why connect nucleoskeleton with cytoskeleton
Makes it possible to deform ncuelus - so immune cels can cross, or other cells
Also mechanisms cancer uses during metastasis = changes in cytoskeleton to get into y tissue
CELL MIGRATION RELYS ON THIS
67
Describe filaments in cells
geenrated by lamins
68
Describe nuclear lamins
Ncuelar lamins = intermediate filaments proteins but diff bc have ncuelar localization sequence= so can get into ncuelus (have nls)
69
Describe how lamins made
2 monomers = associate to dimers
Head to tail association of dimers = make POLYMERS
Then higher order structures
70
Why is lamin structure good
Supports ncuelar structure - prevent form collapsing but issue during mitosis bc want to Dissameble nucleus
71
Describe what happens to nuclear lamins in mitosis
Phosphorylation of head and tail domains = during mitosis = large filaments fall apart
At end of mitosis =regenerate filaments by dephosphorylation
72
What controls filament formation of lamins
Phosphorylation
73
Describe lamin a vs b for mitosis
Lamin a= soluble during mitosis
Lamin b = stays associated with memrvane during mitosis
74
Where are lamins in cell
At nuclear periphery
And in ncuelus interior
75
Describe lamin A
One gene - LMNA, geenrates diff spliced variants lamin a
Developmentally controlled, predominantly in differentiated cells
Some variants lamin a = modified at c term, only some, transient
76
Describe Lamin B
2 genes = LMNB1 LMNB2
ALL lamin b proteins associated with membrane bc modified at c term
Cells contain at least one b type lamin always
77
DESCRIBE MATURE LAMIN A PROTEINS structure
NLS
Long c term
Can cleave = p2 p1 (caax motif = cysteine, any aliphatic aa x 2, any aa)
2 cleavage sites
78
Describe lamin c2 protein structure
NLS
No cleavage sites/motifs
Isoform lamin a
79
Describe lamin b protein structure
NLS
P1 = cleaves right at caax
80
What is isoprenylation
Can modify c term with farnesyl moiety
3 isoprenes = put onto cysteine at c term
81
Describe steps of synthesis and processing lamin a = name
Protein generated
Isoprenylation
First proteolytic cleavage
Second proteolytic cleavage
82
Describe steps of synthesis and processing lamin a = protein generated
Initial translation prodcuct lamin a
Protein 664 aas
Contains c term caax motif
83
Describe steps of synthesis and processing lamin a =isoprenylation
Modify cysteine
Covalent modifcation of c in caax motif
= now sticky, want to associate with membrane
84
Describe steps of synthesis and processing lamin a =1st proteolytic cleage
C term 3 residues cleaved off - at p1, remove aax
Then carboxymethylate cysteine
85
Describe steps of synthesis and processing lamin a =2nd proteolytic cleavage
Removal of modified c and additional resides at c term end - p2
= remove cysteine
86
Describe synthesis and processing lamin a image
Farnesylation - farnesyl transferase
Hydrophobic Moiety on cysteine
Then first cleavage, zmpste24 then carboxymethylate then second cleavage = zmpste24
= WHOEL C TERM GONE
87
Why do we isoprenylate lamin a and process it
HAS Hydrophobic moiety - is associated with membrane = interacts with it
But after mods = free to interact and associate with other lamins = allows Lamina to be in diff locations and have diff functions
88
Name binding partners for a type lamins
Nuclear architecture proteins - of inner nuclear membrane, laps (lamin associated proteins, emerins)
Chromatin organization = his tones, inside ncuelus, promote signalling proteins
Gene regulation = retinoblastoma protein, srebp1
Signalling - pkcalpha, 12-lipoxygenase
89
Describe srebp1
Steroid regulated element binding protein = regulated steroid synthesis - tf
Interacts with lamin b receptor (LBR)
Controls genes relevatnt to lipid production
90
Is a lamin a/c ko mouse alive
Yarrrrrr
Can compensate for lamin a functions with lamin b - a bit
= but mouse lives to 3 weeks then dies, mostly of heart problems
91
Why lamins important
Large number of lamin mutations = associated with human parthenogenesis
368 pathogenic and 239 likely pathogenic clinical varients for LMNA gene
92
Describe mutations in lamin a
All over lamin a gene
Many disease associated wirh it
93
Describe lamin ko vs heterozygous mutant
Cells ok - lamin b compensates
But if one mutant = cells look v bad, can be worse than Ko at cellular basis
94
Describe Hutchinson Gilford progeria
Premature ageing disease, die before 20y/o
Cannot cleave at p2 after carboxymethylation = end up wirh progeria
Mutant lamin a= retains hydrophobic moiety at c term and keeps c term
95
Name types of diseases caused by lamin mutations
Diseases of striated muscle
Peripheral neuropathy - nerve cells
Lipodystrophy syndromes - fat cells
Accelerated ageing disorders
96
Name diseases of striated muscle - lamin a mutation
Autosomal dominant Emery–Dreifuss muscular dystrophy Autosomal recessive Emery–Dreifuss muscular dystrophy Autosomal dominant cardiomyopathy dilated 1A
Autosomal dominant limb girdle muscular dystrophy type 1B
97
Name diseases of peripheral neuropathy - lamin a mutation
Autosomal recessive Charcot–Marie–Tooth disorder type 2B1
98
Name diseases of lipodystrophy syndromes - lamin a mutation
Autosomal dominant Dunnigan-type familial partial lipodystrophy Autosomal dominant lipoatrophy with diabetes, hepatic steatosis, hypertrophic cardiomyopathy and leukomelanodermic papules Autosomal recessive mandibuloacral dysplasia
99
Describe accelerated aging disorders - lamin a mutation
Autosomal dominant atypical Werner Syndrome
Autosomal dominant Hutchinson–Gilford progeria syndrome***
Autosomal dominant restrictive dermopathy lethal
100
Why so many diseases from lamin a mutation
Lamin a supports nuclear organization/structure, lamin a gets weak - since heart cells contract much = damage quick and die = mechanical problem
Effects pleiotropic - affect multiple pathways like dna repair, cell division, vision, oxidative stress - causes premature ageing
101
Describe the 2 major models of how lamin a mutations can alter physiology of cells
1= Lamin affections mechanical properties of ncuelus - like heart msucle cells
2 = Gene expression hypothesis - cell type specific changes in gene expression profiles
= lamin a modulates expression fo certain genes - involved in lipid metabolism
Effect genes needed for nerve cells to properly develop or function
