Nucleus Flashcards
What is the nucleus?
Largest organelle (typically one nucleus per cell)
Nuclear size varies from cell-to-cell and between organisms
Usually determined by cell size (cytoplasmic volume)
Increases during development and in cancer cells
In cancer cells, the nucleus swells and becomes larger
Only Eukaryotes have a nucleus (prokaryotes have nucleoid)
The control centre for the cell
Larger nucleus, the larger the cell
What is special about eukaryotes?
Eukaryotes possess a membrane-bound nucleus
What consists of the outer nucleus?
Nuclear envelope: the boundary between cytoplasm and nucleus
Nuclear pores: ‘doorways’ in the nuclear envelope; regulate transport in/out of the nucleus
What consists of the inner nucleus?
Nucleolus: site of ribosome synthesis
Nuclear matrix: Fibrillar protein ‘mesh’ (network); serves in structural support & chromatin scaffold
Nucleoplasm: ordered architecture; the site where chromatin is found and where RNA processing takes place (DNA is found here)
Prokaryotes possess a ‘region’ (nucleoid) where the chromosome is located, also have less DNA, less DNA packaging, and little or no RNA processing
What is the eukaryotic differentiation of the nucleus from prokaryotes?
Larger, structurally & functionally more complex interiors
Possess single and double membrane-bound organelles
Cellular “compartmentalization” allows for larger size and segregation and organization of specific cellular functions
Compartmentalization: certain areas contain specific organelles, which each perform a specific function
Each organelle contains both unique and common factors for the functioning (e.g. metabolism) and their biogenesis (formation), maintenance, and turnover
What is the nucleus responsible for?
1) Compartmentalization of the cellular genome and its activities
Site of DNA replication, transcription, RNA processing
Sit where translation components (ribosomes, mRNAs, tRNAs) are synthesized
2) Coordination of cellular activities
Control of metabolism, protein synthesis, reproduction (cell division), the control centre
What is the nucleus gene expression?
Separation of the cytoplasm from the genome allows for unique (spatial & temporal) regulation of gene expression in eukaryotes
Prokaryotes: mRNAs translated while transcription is usually still in progress
Eukaryotes: mRNAs undergo post-transcriptional processing (splicing) before being transported out of the nucleus and then translated into the cytoplasm or at the ER
Additional control of gene expression in eukaryotes
Nucleus (nuclear envelope) limits access of transcription factors (proteins) from the cytoplasm to the genome
What is the internal organization of nuclear structure?
- Nucleoplasm
- Fluid-filled interior of the nucleus: highly organized
- Consists of >30 specialized regions (‘subdomains’) that participate in specific functions (note: nuclear subdomains are not membrane bound)
- Nucleus
- Most obvious nuclear subdomain – irregular shaped, dense, and granular in appearance
- Size and number (1-5 nucleoli) depend on the metabolic activity of the cell (cellular activity, protein synthesis, size/number of nuclei) – more active, more nuclei
- Function in producing ribosomes
- Site of ribosomal DNA (rDNA) gene transcription, rRNA processing, and initial stages of ribosomal subunit (rRNA + protein) assembly
- Final assembly of ribosomes (used for protein synthesis) occurs in the cytoplasm
- Chromosomes during interphase are organized into discrete subdomains (territories) within the nucleus
- Location of a gene is often related to its activity
- Most actively transcribed genes are found at the periphery of a chromosomal subdomain
- Chromosomes are also organized into sub-domains
- Interchromosomal channels – regions between subdomains that serve as barriers to prevent unwanted DNA-DNA and/or DNA-protein interactions
- This is a regulatory mechanism
- Prevents unwanted mixing between genes
- Organized is strictly controlled by the nucleus
- Chromosomal subdomains
- Active genes (euchromatin) from different subdomains (or from different regions of the same chromosome) extended into interchromosomal channels to form transcription factories where transcription factories are concentrated
- Interchromosomal Interactions: “kissing chromosomes”
- Gene regulatory regions from one chromosome can active a gene(s) on another chromosome
- Nuclear Speckles
- Subdomains (appear as ‘speckles’ via fluorescence microscopy) –> mRNA processing here, where mRNA splicing factors are concentrated (where pre-mRNA processing occurs)
- Often located in interchromosomal channels next to transcription factories
- Numerous and highly dynamic – ‘speckles’ (>50 in number) can move quickly and grow/shrink depending on the needs of the cell
- Nuclear Matrix
- Insoluble fibrillar-like protein network (‘mesh’) distributed throughout the nucleoplasm
- Analogous to the cytoskeleton network in the cytoplasm
- Composed of three major filament systems: microtubules, actin microfilaments & intermediate filaments
- Serves a structural role - maintains the overall shape of the nucleus
- Serves as a ‘scaffold’ – responsible for organizing nuclear subdomains and anchoring protein factors (e.g., proteins involved in DNA replication, transcription, RNA processing, etc.)
- Very little is known about the composition and assembly/disassembly of the nuclear matrix
- Nuclear Envelope
- Separates the contents (genome) of the nucleus from the surrounding cytoplasm
- Serves as a barrier – requires the regulated passage of molecules (RNA and proteins) between the nucleus and cytoplasm
- Establishes the unique composition of the nucleus (compared to the cytoplasm) and spatially regulates gene expression
- Provides the structural framework for the nucleus
- Composed of the three main parts:
- Nuclear membranes
- Nuclear lamina
- Nuclear pore complex (allow movement)
- Nuclear Membranes
- Inner and outer nuclear membranes – two concentric membranes (phospholipid bilayers) arranged in parallel
- Inner and outer membranes separated by the nuclear envelope lumen (10-50 nm diameter)
- Serves as barriers to the passage of ions, solutes, and macromolecules between the nucleus and cytoplasm
- Outer Nuclear Membrane
- Outer nuclear membrane is continuous with the rough endoplasmic reticulum (ER) lumen
- Ribosomes attached to cytoplasmic surface of the outer membrane (functionally similar to RER)
- Nuclear envelope lumen is continuous with the ER lumen
- Inner Nuclear Membrane
- Unique protein composition (functionally distinct from outer membrane)
- Outer and inner membrane joined at the nuclear pore complexes: connected here
- Nuclear Lamina
- Located on the inner surface (nucleoplasm side) of the nuclear inner membrane
- Network (‘mesh’) of long, filament-like proteins
- ABC nuclear lamins – evolutionary related to proteins that form intermediate filaments in the cytoskeleton network
- Provides mechanical support to nuclear envelope (binds to nuclear inner membrane integral proteins)
- Serves as scaffold for attachment of chromatin and nuclear matrix to the nuclear envelope
- Allows IM to bind to lamina
- Allows nuclear matrix to bind to lamina
- Nuclear Lamina
- Mutations in LAMIN genes responsible for several human diseases
- Hutchison- Gilford progeria syndrome:
- Rare, characterized by premature aging in children (hair loss, wrinkles, artery damage) – death by early adolescence
- Due to a point mutation (sporadic – occurs during in embryo development) in LAMIN A gene (LMNA) leading to a truncated lamin protein
- Results in destabilization/breakdown of nuclear lamina – causes aberrant changes in nuclear envelope morphology and function
- Recently, promising advances using CRISPR/Cas9 genome editing based (gene) therapy in mice (treating the disease? Gene targeting?)
- Nuclear Pore Complex (NPC)
- Channels (‘doorways’) in the nuclear envelope
- Responsible for the regulated trafficking (import & export) of all substances between the nucleus and cytoplasm
- Small, polar molecules (nucleotides for DNA/RNA synthesis)
- RNAs – mRNA, tRNA, rRNA
- Proteins – transcription factors, RNA-binding proteins, ribosomal (subunit) proteins, and cyclins
- Typically, 3000-4000 per nucleus – number of NPCs related to nuclear activity – more active, more doorways
- Large, highly complex structure (30x > ribosome)
- Nuclear Pore Complex (NPC): Continuation
- Composed of approximately 40 different proteins – nucleoporins (‘Nups’)
- Highly conserved among all eukaryotes
- Include both integral and peripheral inner and outer nuclear membrane proteins
- Several Nups related to COPII proteins involved in vesicle formation at the ER
- Suggests common evolutionary origin – both Nups and COPII proteins function to deform (highly curve) membranes
- Overall structure of NPC: 8-fold symmetrical structure organized around a large, central aqueous channel
- Nuclear Pore Complex (NPC): Continuation
- Consists of several parts:
- Central scaffold – composed of (integral-bound/trans) membrane nucleoporins
- Anchors NPC to the nuclear envelope
- Forms an aqueous central channel (20-40 nm wide pore)
- Nuclear Pore Complex (NPC): Continuation
- Inner surface of the channel lined by ‘filament-like’ Nups – FG nucleoporins
- Possess an unusual amino acid composition
- Mostly hydrophobic polypeptides with short repeats of hydrophobic domains enriched in phenylalanine’s and glycine’s (FG domains): contain many domains
- FG Nups possess a unique, highly disordered secondary structure
- Extended/flexible organization that fills central channel
- FG Nucleoporins
- FG domains extend into central channel
- Form a ‘mesh’ (sieve like gel) that limits the diffusion of macromolecules larger that approx. 40 kDa
- Small molecules can move freely through the NPC in either direction (e.g. Nucleotides for DNA & RNA synthesis)
- Molecules >40 kDa unable to pass through the NPC freely –> active process to move through
- RNA and most proteins must be selectively imported/exported by an active process
- Size-exclusion limit for the NPC
- Based on studies using microinjected gold particles of varying sizes and coated with a nuclear protein
- Determine size-exclusion limit of NPC: Microinjection of nuclear protein-coated gold particles into individual mammalian cultured cells.
- Nuclear Pore Complex (NPC): y complexes
- Includes cytoplasmic and nuclear rings
- Composed of structural nups
- Located on the cytoplasmic and nuclear (nucleoplasm) side of the NPC, respectively
- Linked to the central scaffold and also the cytoplasmic filaments or the nuclear basket
- Cytoplasmic Filaments
- Long filament shaped (structural) Nups that extend into the cytoplasm
- Involved in nuclear receptor-cargo protein recognition and import from the cytoplasm
- Nuclear Basket
- ‘basket like’ structure (also made of structural Nups) located on nuclear nucleoplasm) side of the NPC
- Involved in nuclear receptor cargo protein import and export to the cytoplasm
- Linked to Y complex/nuclear ring
Nucleocytoplasmic transport via the NPC
• Among the most congested bi-directional trafficking pathways in the cell
• Includes a variety of cytoplasm- to- nucleus (‘import’) and nucleus- to- cytoplasm (‘export’) trafficking pathways
• All proteins required for DNA replication, transcription, splicing, ribosome assembly, chromatin packing (histones), must be imported into the nucleus from the cytoplasm
• All RNA (mRNA, tRNA, rRNA), partially assembled ribosomes (required for protein synthesis in the cytoplasm), and some proteins, must be exported out of the nucleus into the cytoplasm
• Molecular mechanism is well understood – requires energy, specific protein receptors, and unique targeting signals
• Cytoplasm to nuclear transport (import)
• Most nuclear- imported proteins (transcription factors, nuclear matrix proteins, histones, lamins, cyclins, etc) contain a nuclear localization signal (NLS)
• Specific stretch/sequence of amino acids that are recognized by nuclear receptor proteins, which serve as a zipcode to mediate targeting of the protein from the cytoplasm to the nucleus
• Several different types of NLSs – based on different (unique) amino acid sequences
What is NLS’s?
- Classic NLS: most common NLS and first to be identified
- Consists of a short stretch of positively- charged (basic) amino acid residues
- KKQRKK in the large T antigen of simian virus 40
- Bipartite NLS: composed of two short stretches of basic amino acids and a 7-10 amino acid long spacer sequence
- KR[PAATKAGQA]KKKK
- Protein can have more than one NLS and NES
- NLS(s) identified in proteins based on mutational analyses
- Definition of an NLS: an amino acid sequence that is both necessary AND sufficient for cytoplasm to nuclear targeting
- Necessary: if the sequence (or a portion of it) is mutated, then the modified protein (loss of function) fails to target to the nucleus (mutant protein is mislocated to the cytoplasm
- Sufficient: if the sequence linked to a non-nuclear (passenger) protein is capable of redirecting the resulting fusion protein (gain of function) to the nucleus)
What is cytoplasm to nucleus transport?
- Characterization of different NLSs led to the identification of factors necessary for the nuclear import of proteins from the cytoplasm
- Including transport receptors, which are mobile proteins responsible for moving (ferrying) protein ‘cargo’ across the nuclear envelope
- Karyoferins: Large family of receptor proteins responsible for moving macromolecules (protein or RNA) either into the nucleus (importins) or out of the nucleus (exportins)
- Protein important into the nucleus is a multi-step process
What are the steps of cytoplasm to nucleus transport?
- Nascent (newly synthesized) NLS-containing ‘cargo’ protein is recognized in the cytoplasm by importin
NLS: indicates that protein is going to nucleus - ‘Cargo’ protein importin receptor complex moves through the cytoplasm, towards the nucleus (via importin’s ability to bind the cytoskeleton).
Step 2 continued:
At the surface of the nucleus, importin ß subunit of cargo-protein importin receptor complex binds to a cytoplasmic filament at the NPC - ‘Cargo’ protein important receptor complex is translocated through central channel of NPC –> this process is regulated and if the protein does not have an NLS, it cannot go through the channel
Translocation: cargo-receptor complex successively interacts with hydrophilic and FG domains of FG Nups in central channel, the interactions ‘dissolve’ (untangle) the FG- domain network and allow for translocation through the central channel. - ‘Cargo’ receptor complex associates with the nuclear basket on the inner surface of the NPC.
‘Cargo’ receptor complex binds to Ran-GTP (via importin ß) resulting in its release from the NPC and disassembly into the nucleoplasm (released in nucleoplasm, indirectly delivered)
Import of the NLS containing ‘cargo’ protein into the nucleus is completed, but the NLS is still attached - Ran-GTP bound importin ß subunit moves back into the cytoplasm due to the [Ran- GTP] gradient
In the cytoplasm, GTP on Ran-GTP is hydrolyzed via the accessory protein of Ran-GAP, and Ran-GDP is released from importin ß
importin ß can be used for another round of nuclear protein important (indirectly because of the concentration gradient)
Ran-GDP released from importin ß moves back into the nucleus due to the [Ran-GDP] gradient
Ran-GDP in the nucleus is converted into Ran-GTP by the accessory protein GEF
Ribosomal subunit proteins and proteins that bind to RNAs (mRNA, tRNA, rRNA), are exported out of the nucleus into the cytoplasm.
Step 5 continued: importin ⍺ binds to exportin and the release of the nuclear imported ‘cargo’ protein exposes a nuclear export signal (NES) in importin ⍺
NES indicates that it needs to be transported out of the nucleus to the cytoplasm
Exportin also binds to other ‘cargo’ proteins due to be exported from the nucleus via their NESs (several types of NESs)
Importin ⍺ (or an NES containing ‘cargo’ protein)- exportin complex binds Ran-GTP (high [Ran-GTP] in the nucleus)
Importin ⍺ exportin Ran GTP complex is transported (via the NPC) into the cytoplasm due to the [Ran-GTP] gradient
In the cytoplasm, GTP on Ran-GTP is hydrolyzed by Ran-GAP
Ran-GDP is released from exportin and release of importin ⍺ or the NES- containing ‘cargo protein:
Importin ⍺: used for another round of import
Ran-GDP moves back to the nucleus due to the Ran-GDP gradient and converted via GEF into Ran- GTP
Exportin: moves back into the nucleus (via importin) for another round of export
[Ran-GTP] nucleus < [Ran- GTP] cytoplasm
[Ran-GTP] nucleus < [Ran- GTP] cytoplasm
What are the types of NES’s?
- There are several types of NESs
- All are both necessary and sufficient for nucleus to cytoplasm targeting
- Most common NES consists of a leucine-rich motif: LxxLxxL
What are some cytoplasm to nucleus transport notes?
- Step 1: importin is a heterodimeric protein
- Consists of two distinct subunits importin ß and importin ⍺
- importin ⍺ subunit binds to the basic residues in the ‘cargo’ protein’s NLS
- Step 2: Cytoskeleton elements serves as ‘highways’ for almost all types of intracellular transport (movement of proteins, RNA, organelles)
- Step 3: Translocation process is not well understood
- Step 4: NLS is not proteolytically cleaved from the cargo protein, unlike most other organelle targeting signals.
- Step 5: See Ran below
- Step 5 continued: karyopherin that mediates nuclear to cytoplasm transport
- NESs: specific stretch/sequence of amino acids that are recognized by exportin and serve as a ‘zipcode’ to mediate targeting of the protein from the nucleus to the cytoplasm
What is Ran?
- Small GTP binding protein
- Protein’s conformation and activity is regulated by GTP binding & hydrolysis
- Exists in two distinct states:
- Ran-GTP (active GTP-bound form)
- Ran-GDP (inactive GDP- bound form)
- A steep concentration gradient of Ran-GTP exists between the nucleus and cytoplasm:
- [Ran-GTP] nucleus > [Ran- GTP] cytoplasm
- Gradient controls movement in and out of nucleus
- [Ran-GTP] gradient between the nucleus and cytoplasm is mediated by two accessory proteins:
- GEF: nuclear protein that promotes the conversion of Ran-GDP to Ran-GTP, and it maintains a high [Ran-GTP] in the nucleus –> promotes the GTPase activity of Ran
- Ran-GAP: cytoplasmic protein that promotes the hydrolysis of Ran-GTP to Ran-GDP, and it maintains low [Ran-GTP] in the cytoplasm
- Ran-GTP gradient determines the directionality of nucleocytoplasmic transport
- GTP hydrolysis provides the energy required for nucleocytoplasmic transport
- A concentration gradient of Ran-GDP exists:
- [Ran-GTP] nucleus < [Ran- GTP] cytoplasm
What is Nucleocytoplasmic transport?
- Some proteins are imported back into the nucleus without an NLS
- Referred to as ‘piggyback’ nuclear protein import
- Nascent protein lacking an NLS binds to an NLS containing protein in the cytoplasm
- Targeting and import of the protein-protein complex into the nucleus is mediated by importin receptors
- Many proteins ‘shuttle’ between the nucleus and cytoplasm
- Participate in both nuclear and cytoplasmic functions
- Often contain both an NLS and NES
- The relative distribution of the protein in either compartment is controlled by the relative strength of its NLS and NES
- NLS > NES = the majority of the protein (at steady state) is localized in the nucleus
- Strength of NLS and NES can be controlled by a post-translational modification
- Phosphorylation of a specific amino acid residue(s) that comprise of and/or adjacent to the targeting signal
- LxxLxxLxx or KKQRKKx x = S/T/Y (can be phosphorylated and no longer detected as an NES or NLS)
