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B3.0 > Organelle Ecology - Organelles without Membranes > Flashcards

Organelle Ecology - Organelles without Membranes Flashcards

1
Q

Structure:

A
  1. nucleolus
  2. P. granules
  3. pyrenoids
  4. carboxysomes
  5. biotech
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2
Q

Nucleolus

A
  • large, discrete nuclear sub-compartment
  • heterochromatin coat
  • ribosome biogenesis
  • size correlates w/ rRNA synthesis, cell growth and metabolism
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3
Q

Components of the nucleolus

A

1) FC
2) DFC
3) GC

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4
Q

fibrillar centre

A
  • FC
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5
Q

dense fibrillar component

A
  • DFC
  • requires FBL interaction with nascent transcript
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6
Q

granular component

A
  • GC
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7
Q

Describe the nucleolar components

A
  • tripartite
  • recognisable, membrane-unbound regions
  • specific protein complements
  • differential staining + EM
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8
Q

NORs

A
  • present on multiple chromosomes
  • rDNA
  • at least 1 nucleoli form around them
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9
Q
  • nucleolar organiser regions
A

NORs

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10
Q

rDNA

A
  • array of tandem repeats
  • intergenic spacers
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11
Q

pre-rRNAs

A
  • transcribed from multiple rRNA repeats
  • needs lots (ribosomes, protein synthesis)
  • e.g. RNAP1: 45S pre-rRNA
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12
Q

RNA components in growing cells

A
  • r: ~80%
  • t: ~15%
  • m: ~5%
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13
Q

rRNP complices

A
  • pre-export
  • 28S, 18S, 5.8S rRNAs
  • processed by snoRNPs
  • create small (40S) and large (80S) subunits + 5S rRNA
  • NPC translocation
  • active in cytoplasm
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14
Q

Sequential processing in each nucleolar component

A
  • FC: pre-rRNA transcription
  • DFC: pre-rRNA splicing
  • GC: large + small subunit assembly
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15
Q

Nucleolar division dynamics

A
  • dissolution + reformation
  • separation and fusion (coalesce)
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16
Q

Isolated nucleoli

A
  • liquid-like properties
  • fuse spontaneously/when compressed
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17
Q

Germinal Vesicles

A
  • GVs
  • can be studied in Xenopus oocytes under actin disruption, nucleoli sedimentation and coalescence
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18
Q

Xenopus oocytes

A

~0.05mm (> somatic nuclei)
- 1-1500 large extrachromosomal nuclei
- LOTS of ribosomes
- cores of tandem rRNA mini chromosomes (gene amplification)

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19
Q

FRAP

A

rapid molecular exchange (tenths of s) w/ surrounding nucleoplasm

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20
Q

Nucleolar proteins

A
  • fibrillarin (FBL)
  • nucleophosmin (NPM1)
  • phase separate in vitro; form condensated lipid droplets
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21
Q

phase separation

A
  • occurs if local viscosity prevents mixing of different regions
  • compartmentalisation and concentration of protein and RNA at specific loci, without membrane barriers
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22
Q

what is phase separation driven by?

A
  • weak multivalent dynamic interactions, repeated molecular domains and proteins w/ intrinsically disordered regions (IDRs)
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23
Q

condensate formation is promoted by

A

bifunctional proteins @ specific genomic loci

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24
Q

Describe the process of condensate formation

A

1) tethering domain binding (specific DNA/RNA/protein sequence)
2) condensate binding domain (multivalent interactions through IDRs; multiple binding molecules)
- as the binding interactions increase, there is a phase transition to condensate

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25
bimolecular nuclear condensates
1) super-enhancers 2) speckles 3) insulation loop anchors 4) polycomb bodies 5) heterochromatin 6) nucleolus
26
The heterochromatin that surrounds the nucleolus
is its self a bimolecular nuclear condensate
27
Nucleolar LLPS
28
FBL
- GAR domain contains IDRs
29
GAR
glycine-Arg-rich
30
NPM1
- GC scaffold - acidic (A1-A3) + basic (B1-2) motifs in IDR - pentamerisation domain
31
oligomerisation domains
- couple w/ substrate binding domains - amplifies interaction valency
32
Nucleolar initiation
1. nucleation promoted by rRNA transcription @ NORs 2. sequential processing 3. radial rRNA expulsion
33
Nucleolar maintenance
- FBL and NMP1 competition limits interaction zones - as rRNA assembles, NPM1 contacts decrease; decreased GC solubility - selective exclusion of fully processed and assembled subunits
34
P-granules
- accumulate @ C. elegans posterior pole post-fertilisation and pro-nuclear migration - asymmetrically inserted - determine germline - contain protein, RNA - can be perceived under super-resolution microscopy
35
C. elegans
- 959 cells - known fate map - v important ACDs
36
Polarisation in C. elegans in
initiated by sperm entry site
37
Describe polarisation in C. elegans
1. sperm entry localises PAR-2 to posterior pole 2. acto-myosin contractions generate cortical flow, spreading the domain 3. PAR-2 and -3 undergo reciprocal inhibition 4. Cdc42-PAR-6-PKC complex maintains polarity @ anterior pole 5. PAR-1 reduces MEX-5 concentration @ posterior pole 6. MEX-5 destabilises P granule formation
38
Acto-myosin
- important in P granule formation - an important ESP, formed by genetic duplication and recombination events
39
What does C. elegans polarisation represent?
a dynamic asymmetric dissolution-condensation model
40
PGL1
- RGG domain protein - assembles liquid phase
41
MEG-3
- IDRs - assembles supporting gel-like phase
42
MEG3/4
- IDRs - mRNA localisation and trapping in the core
43
pyrenoid
- part of CCM in many algae - e.g. Chlamydomonas - RUBISCO localisation: important - visible under L+EM - unbound, discrete - LLPS prevents back-diffusion
44
Carbonic anhydrases
- CO2 + H2O -> HCO3- + H+ - bicarbonate is charged and can be transported to the thylakoids - decreases photorespiration
45
Pyrenoid inheritance
- divide terminally to chloroplastic division - splits into 2x droplets - occasionally asymmetrically inherited - must form de novo, from the aggregation of new droplets
46
RUBISCO packing
- consistent with short-range liquid-like organisation (NOT a crystalline array) - under rapid freeze EM tomography, which allows localisation of all complices
47
FRAP
- fluorescently-tagged RUBISCO in live and fixed cells - bleach 1/2 pyrenoid and follow recovery - RUBISCO mixes w/ live pyrenoids in ~20s
48
Additional pyrenoid interactions?
protein tethering to the starch envelope stops CO2 diffusing out, ensuring functionality
49
Cyanobacteria carboxysomes
- RUBSICO interacts w/ multivalent proteins to form condensates
50
homology between pyrenoids and carboxysomes
- EPYC1 is homologous to CCMM - both have IDRs - form LLPS in vitro
51
EPYC1
essential pyrenoid component 1
52
CCMM
carbon contracting mechanism M
53
Algal RUBISCO in heterologous expression in higher eukaryotes
forms LLPS
54
Evidence of LLPS
- not much! - mostly qualitative, and indirect
55
Phase-separation
- transient, low-affinity, hydrophobic interactions between MVPs / IDPs - macromolecules are attracted to the same compartments via homotypic interactions
56
MVPs
multivalent proteins
57
IDPs
intrinsically disordered proteins
58
SSIs - an alternative explanation
- site-specific interactions - low kDas (irreversible) - high kDas (regulated interactions; easily adjustable to non-equilibrium conditions in active processes) - rapidly modulated through PTMs - no change. in binder conc.
59
biotechnological applications
- concentrating within compartments - sequesters toxins - protects proteins from degradation
60
Artificial LLPS in plants
i) make construct +/- IDR (+NLS) ii) changes conc = yes - at a certain concentration, the system will flip to LLPS; threshold
61
What happens at the LLPS threshold?
concentration remains constant whilst volume increases
62
metabolic engineering
- make biodegradable polymers (e.g. pre-plastics)
63
PHB
2 acetyl CoA -(beta-ketothiolase)-> acetoacetyl CoA -(acetoacetyl CoA reductase)-> 3-hydroxybutyryl-coA -(PHB synthase)-> PHB
64
If you engineer PHB in LLPS, what happens?
- it works! - 4x increase compared to wt
65
Potential explanations for how it works?
1) enzyme degradation protection? 2) product degradation protection? 3) decreased diffusion?

B3.0 (44 decks)

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