Nuclear lamins, nuclear pores, biocondensates Flashcards
What is phase separation in cells?
- Phase separation is the ability of biomolecules (proteins, RNA, etc.) to form distinct, membrane-less compartments within cells.
- It occurs through liquid-liquid phase separation (LLPS), where molecules organize into droplet-like structures, similar to how oil and water separate.
- These compartments concentrate specific biomolecules to regulate biological processes, such as RNA processing and stress response.
What is the function of membrane-bound organelles?
- ## Membrane-bound organelles (like the nucleus) separate the internal environment from the cytoplasm and allow for specialized functions and controlled chemical reactions in microenvironments.
What role does phase separation play in the nucleus?
- In the nucleus, phase separation organizes nuclear substrates and substructures, including the nucleolus, which is responsible for ribosome production.
How is the nucleus organized?
(1) Nuclear Envelope
- surrounds nucleus and acts as a barrier.
- has 2 membranes; inner and outer membrane, the outer connects to the ER. There is a perinuclear space between the two membranes.
- has nuclear pore complexes (NPCs); large openings that control what enters and leaves the nucleus, these pores use phase seperation to regulate movement of molecules like RNA and proteins.
(2) Nuclear Lamina
- supportive meshwork of proteins beneath inner membrane (gives nucleus its shape and helps anchor chromatin)
(3) Nuclear Interior
a) nucleolus (site of ribosome production - forms through phase seperation)
b) chromatin (phase seperation helps regulate gene expression/transcription)
c) nuclear bodies (form through phase seperation and help with RNA processing)
d) nuclear matrix (structural framework that helps organize chromatin and nuclear processes)
What is the function of nucleolus (nucleoli)?
- produces and processes 47S ribosomal RNA
- assemble ribosomal subunits
- assemble signal recognition particle (SRP)
- regulate apoptosis
- control cell cycle progression
- regulate p53 function
- control telomerase function
- regulate stress response
- play a role in replication of many viruses
- nucleolus divided into different sub-compartments formed through phase seperation. (granule component, GC dense fibrillar component, FC, RNA-Pol I)
- formed through LLPS, inside them behave like liquid droplets, allowing dynamic molecular exchange.
- FBL in DFC & NPM in GC-important proteins for ribosome production (proteins mix to form droplets)
- directly related to disease so we care about them a lot.
Ribosomal Biogenesis (combining rRNA with ribosomal proteins)
(1) rRNA transcription
- 28 rRNA, 18 rRNA, 5.8 rRNA transcribed inside nucleolus.
- 5S rRNA is transcribed outside the nucleolus in the nucleus.
(2) Assembly of rRNA and Ribosomal proteins.
- ribosomal proteins are synthesized in the cytoplasm.
- they enter the nucleus and combine with rRNA to form large and small ribosomal subunits.
- large: 28s, 5.8s,ribosomal proteins.
- small 18s, ribosomal proteins.
- these are assembled in nucleolus, then subunits are exported back to cytoplasm where they combine to form functional ribosomes.
What is the connection between cancer and nucleoli?
- cancer cells often have enlarged nucleoli due to increased ribosome production. this correlates with increased drug sensitivity. (rRNA synthesis inhibitors)
- higher the rate of ribosome production, more effective is the killing of cancer cells with drugs that target the nucleolus.
Why do proliferating cells assemble more ribosomes?
(1) Increased protein production (need more growth, cell division, and new structures)
(2) Support for rapid cell division.
(3) Efficient protein synthesis.
What are characteristics of the NPC?
- 8-fold symmetry
- composed of around 1000 proteins
- 35 distinct proteins
- FXF repeats (amino acid sequence that binds proteins involved in nuclear import)
- GLFG repeats (help bind RNA and anchoring proteins, play a role in RNA export from the nucleus)
- molecules larger than 40kDa = active transport
- small molecules = passive transport
What are nuclear envelope proteins?
- Inner membrane:
(1) LBR (lamin-B receptor) - anchors chromatin and lamina to inner membrane.
(2) LAP2 - nuclear envelope organization and chromatin binding.
there are also transmembrane domains and chromatin/lamina interaction domains. (interactions effect gene expression)
What is function of nuclear lamina?
- The nuclear lamina is a fibrous network of proteins located just beneath the inner membrane of the nuclear envelope.
- Linked to the actin cytoskeleton, helping maintain nuclear shape and positioning in the cell.
- assist in mechanical stability and cell signalling.
Lamins are proteins that form a network inside the nucleus, nuclear lamina, structural support/organization.
Lamins first form dimers, assemble head to tail, forming filaments, that create a scaffold around the nucleus.
What is the function of SUN and KASH domain proteins?
- link the nuclear interior to the cytoplasm.
- LINC = linker of nucleoskeleton and cytoskeleton.
What are physiologically relevant reasons for connection cytoskeleton with nucleoskeleton?
(1) Immune cell migration.
- allows nucleus to deform and squeeze through tight spaces, crucial for immune cells to move through tissues during immune responses.
(2) Cancer metastasis.
- similar mechanism to move and spread to other parts of the body. Migrate through tissue barriers, cancer cells and reshape nucleus, aiding in metastasis of tumours.
How are lamins regulated during mitosis (cell division)?
During mitosis, lamins are phosphorylated, causing them to disassemble and allowing the nucleus to break down. This facilitates the process of cell division.
At the end of mitosis, dephosphorylation occurs, leading to the reassembly of the lamins and the reformation of the nuclear envelope.
Lamin A becomes soluble during mitosis, which helps in the breakdown of the nuclear structure.
Lamin B and C remain attached to the nuclear membrane throughout mitosis, ensuring structural integrity and anchoring of the envelope.
Lamin A and Lamin C are both produced from the LMNA gene, not LMNBI and LMNB2. Lamin B is produced from the LMNB1 and LMNB2 genes.
Lamin A, B, and C can all undergo alternative splicing, leading to variants with different functions and properties..
What is Lamin A and its structure?
Lamin A is a 664 amino acid protein with a C-terminal CAAX motif, which is involved in isoprenylation (a covalent modification).
What does the Lamin A gene produce?
The Lamin A gene produces spliced variants, including Lamin A and Lamin C.
Lamin A Processing Steps
(1) First cleavage involves proteolytic processing of the C-terminal CAAX motif. This removes the CAAX motif and includes carboxymethylation (P1).
(2) Second cleavage involves removing the modified C-terminal and additional residues at the C-terminal end (P2).
What are the binding partners for A-type lamins (lamin A and lamin C)?
Proteins that play a role in:
- nuclear architecture (proteins of inner membrane, CAPs, emerin)
- chromatin organization (histones)
- gene regulation (retinoblastoma protein, SREBP1)
- Signalling
Is a lamin A/C KO mouse alive?
- survive 3 weeks, die from heart failure.
- defects in muscle structure and function.
- can differentiate cells, but fail to maintain normal function, especially in tissues like the heart, where Lamin A is essential for normal tissue/function.
- mutations in LMNA (gene encoding lamin A) can cause severe nuclear defects.
- mutant in lamin A can be more damaging than absense of lamin A itself.
What causes Progeria (Hutchison - Gilford Syndrome)
- caused by mutation in the C-terminal of LMNA gene, leads to production of abnormal protein called progerin.
- progerin has a hydrophobic region which causes premature aging in cells -> you would have premature aging.
- patients usually die before 20 years old.
- Lamin A/C proteins have longer lifetime in tissues like aorta and heart and fat tissue. These proteins are more insoluble in cardiovascular tissue.
- Progerin has EVEN longer lifetime, accumulates in tissues, further structural damage. Correlates with decrease rate of protein turnover, body has hard time breaking down and replacing damaged proteins.
Why do different LMNA mutations cause different diseases?
(1) DNA repair defects.
(2) Pathway disruptions.
(3) Cell-specific type effects.
BUT
all diseases related to mutations in LMNA gene, which encodes lamin A/C protein, involves defects in nuclear envelope and nuclear structure, leads to many pathological efffects, muscle degeneration, cardiovascular proteins, fat loss, premature aging.
