the nucleus - lecture 12

morphology of the nucleus

• nuclear pore complex

• nucleolus

• chromatin

• lamina

phase-separated compartments coexisting in the nucleoplasm

• PML body

• stress granule

• autophagosome

• sHSP compartment

• IPOD

• JUNQ

• p62 sequestosome

• nucleolus

• nuclear stress granules

nucleoli are membraneless sub-compartments of the nucleus

→ 1 – 5 nucleoli per nucleus

→ > 500 different proteins/nucleolus

→ location where ribosomal DNA (rDNA) is encoded in the genome

→ rDNA is transcribed into ribosomal RNA (rRNA)

→ assemble with r-proteins into mature ribosomes

→ a phase-separated structure with distinct layers

→ RNA required for assembly of the SRP is manufactured in nucleoli as well!

3 distinct zones exist within the nucleoli

→ FC: fibrillar center (transcription of rDNA)

→ DFC: dense fibrillar components (rRNA processing)

→ GC: granular components (ribosome assembly, storage for unfolded proteins)

nucleoli are:

ribosome factories

Gated access to the nucleus: the nuclear pore complex (NPC)

→ two bilayers separate the nucleoplasm from cytosol

→ nuclear pore complex (NPC): spans both membranes for transport between cytoplasm and nucleoplasm

→ proteins < 40 kDa: can diffuse through NPC

→ proteins > 40 kDa: ‣ require a nuclear localization or nuclear export signal

‣ nuclear transport receptors

‣ Ran GTPases

‣ Ran GEFs and GAPs

→ mRNPs are transported by a Ran-independent pathway

nucleoporins stabilize pores in the nuclear envelope:

→ FG-nucleoporins, structural nucleoporins, and membrane nucleoporins are important building blocks of the NPC

a gel-like condensate fills the pore:

→ 16 copies of the Y-complex (8 associated with each membrane, form a ring)

→ FG-repeats are extended disordered structures that have interspersed hydrophilic regions

→ a gel-like condensate that allows diffusion of small molecules but blocks unchaperoned translocation of proteins > 40 kDa

nuclear localization signals (NLSs) direct proteins to the cell nucleus

→ NLSs do not have a strict motif but are rich in basic amino acids

→ 7-residue NLS from SV40 T-antigen: P-K-K-K-R-K-V

nuclear import mechanism - cytoplasm

importin (soluble nuclear transport receptor) binds an NLS of a cargo protein to form an

importin-cargo complex

• importin-cargo complex diffuses through the NPC by transiently interacting with FG-

nucleoporins.

nuclear import mechanism - nucleoplasm

• Ran-GDP activated by GEF, releases GDP and binds GTP

• Ran-GTP binding to the importin causes importin conformational change that releases

the NLS-cargo protein

nuclear import mechanisms - system recycling

• importin-Ran-GTP complex is transported back to the cytoplasm

• GAP associated with the cytoplasmic filaments of the NPC stimulates Ran hydrolysis of its

bound GTP

• Ran-GDP conformational change releases importin (diffuses into nucleus for new round)

what insures direction of nuclear import?

localization of GEF in nucleus and GAP in cytoplasm

Nuclear export mechanism 1/2: Ran-dependent, nuclear export signals (NESs)

→ NESs do not have a strict motif but are rich in hydrophobic amino acids

→ NES from HIV-1: L-X-X-X-L-X-X-L-X-L

nucleoplasm - ran-dependent export

‣ exportin 1 binds to form a complex with an NES-cargo protein and Ran-GTP

‣ complex diffuses through an NPC via transient interactions with FG-repeats in FG-

nucleoporins

cytoplasm - ran-dependent export

‣ Ran-GAP associated with the NPC cytoplasmic filaments stimulates Ran-GTP hydrolysis to Ran-GDP

‣ Ran-GDP conformational change releases NES-containing cargo protein into the cytosol

recycling system - ran-dependent export

‣ exportin 1 and Ran-GDP are transported back into the nucleus

‣ Ran-GEF in the nucleoplasm converts Ran-GDP to Ran-GTP

nucleoplasm - ran-independent export

‣ heterodimeric NXF1/NXT1 nuclear export receptor complex binds to mRNA-protein

complexes (mRNPs)

‣ complex diffuses through NPC by transiently interacting with FG nucleoporins

cytoplasm - ran-independent export

RNA helicase (Dbp5) located on the cytoplasmic side of the NPC uses ATP energy to remove NXF1 and NXT1 from the mRNA

recycling system - ran-independent export

Ran-GDP dependent import process recycles free NXF1 and NXT1 proteins back

into the nucleus

what reinforces the inner membrane of the nucleus?

lamina

the way we picture chromosomes:

→ humans have 23 pairs of chromosomes

→ 22 autosomes, and one pair of sex chromosomes (X and Y)

→ karyograms are prepared in mitotic cells, when chromosomes are maximally condensed

what interphase chromosomes look like in the nucleus:

→ interphase nuclear chromatin is organized into distinct, non-overlapping territories!

structural organization of chromosomes:

→ each chromosome consists of a single DNA molecule (up to 280 Mb)

→ organized into increasing levels of compaction from nucleosomes to higher order chromatin by histone and non-histone proteins

(compaction ratio 1:100,000)

→ compacted DNA can be accessed for transcription, replication, repair

→ only 1.5% of human DNA encodes proteins and functional RNAs

→ remainder: regulatory sequences that control gene expression and introns

→ 45% of human DNA is derived from mobile DNA elements (genetic symbionts that have contributed to evolution of the genome)

structure of the nucleosome:

→ nucleosome: ~147 bp of DNA wound around octamer protein core containing two copies each of histones H2A, H2B, H3, and H4

→ histone surface has positive charges hold the negatively charged DNA

→ ~147 bp of DNA wrapped one and two-thirds turns around the histone core

name for arrangement of nucleosomes in chromatin:

beads-on-a-string

eachromatic and heterochromatic regions:

→ chromatin is composed of structurally disordered chains

→ 24-nm nucleosomes, 5-nm linkers

→ loosely-packed chromatin: euchromatin

→ densely-packed chromatin: heterochromatin

the SMC complex (cohesin) condenses chromatin:

→ SMC complex ‘clamps’ chromatin strands together

→ formed by the coiled-coil proteins Smc2, Smc4, and kleisin

PTMs control compactin of chromatin

→ chromatin function is controlled by post-translational modifications (PTMs) of histone proteins

→ acetylation, methylation, phosphorylation, ubiquitinylation

Histone acetylases, histone deacetylases, and histone deacetylase inhibitors (HDACi’s)

HDACi’s are epigenetic regulators that increase transcription, may reverse age-related epigenetic changes, increase cellular plasticity, and much more

condensed subcompartments within chromosomal territories:

spatial/temporal control of chromatin organization via LLPS?