Biol110-10-Lecture 6-Nucleoplasm

Biol110-10-Lecture 6-Nucleoplasm - The cell nucleus• ...

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Unformatted text preview: The cell nucleus•  Two bilayers •  Held in place by a scaffold •  Perforated by nuclear pores Transmission electron micrograph •  The nucleus protects the cell’s DNA and allows sophisticated gene regulation • It also uncouples transcription from translation No assigned reading for next class There are many nuclear subcompartments- General features: • not membrane enclosed • each is marked by its specific content of proteins, and some have unknown functions • very dynamic aggregates that constantly capture and release their specific components • purpose: to increase efficiency of nuclear processes by concentrating specific enzymatic activities Biomarker for breast cancer Scaffold for the NE and heterochromatin Promyelocitic leukemia Store RNA splicing factors The nucleolus is the ribosome factory of the cellThe most prominent structure in the nucleoplasm under the microscope Nucleoli are dynamic Nucleus Nucleolus (usually 2-3 per nucleus) Nucleolar targeting signals direct proteins to the nucleolus Nucleolus substructure Cajal bodiesCajal bodies are highly dynamic structures of 0.2 - 1.0 µM in diameter. The average nucleus contains 1-10 of these bodies. Cajal bodies are involved in snRNP biogenesis and in the trafficking of snoRNPs and snRNPs, which appear to move through the Cajal body en route to nucleoli or splicing speckles (respectively). Fibrillarin in Nucleoli Inter-chromatin and CBs granules- All four combined CBs first observed by Ramon y Cajal in 1903 Chromatin Gems Origins of the nuclear envelope- Scanning electron micrograph of freeze fractured cell transmission electron micrograph Structure and function of the nuclear laminaThe Nuclear lamina is a scaffold-like network of protein filaments surrounding the inner-nuclear periphery. It is made of intermediate filament proteins, lamin A/C and B, which together form the meshwork shown right underneath the membrane. Functions for the nuclear lamina: •  maintenance of nuclear shape •  spatial organization of nuclear pores within the nuclear membrane •  regulation of transcription (silencing) •  anchoring of interphase heterochromatin •  communication to the cytoplasm via Sun-Nesprin bridges Scanning electron micrograph of a freeze dried & metal shadowed nucleoplasmic face of a Xenopus oocyte. Lamins: the building block of the nuclear laminaThe lamins are coiled-coil structures that contain a small N-terminal head followed by a long rod-like domain (a coiled-coil) and a C-terminal globular tail. Lamins form parallel coiled-coil dimers, which in turn form polymers with other lamin dimers in an anti-parallel manner (head-to-tail). Mutations in the gene encoding for Lamin A are associated with forms of muscular dystrophy, progeria, cardiomyopathy, and many many other human diseases Cut-away diagram of the nuclear envelope- Known lamina-associated and nuclear envelope proteins Sun-Nesprin connections The endoplasmic reticulum membrane is contiguous with the ONM (outer nuclear membrane). Nuclear pore are points at which the INM (inner-nuclear membrane) and the ONM connect Nuclear pore complexes are protein assemblies that gate nuclear pores The location of chromosomes is fixed in the nucleoplasm- The nuclear lamina participates in gene silencing and activation- Silenced gene (sequestered) Activated gene (exposed) Genes close to centromeres and telomeres are silenced and often at the nuclear periphery Breakdown and reformation of the nuclear envelope during mitosis- Study of nuclear envelope assembly using Xenopus extracts- The key role of Ran-GTP ‘clouds’ as a chromatin & nucleoplasm landmark in cellsThe Ran GTPase, its deactivating enzyme (RanGAP), and its activating enzyme (RanGEF) Nucleocytoplasmic transport Mitotic spindle assembly Nuclear envelope assembly RanGTP governs transport RanGTP is needed for spindle assembly RanGTP is needed for vesicle fusion Ran-GTP functions by binding to karyopherin/importin beta receptors to modify their structure and functionKaryopherin/importin beta Ran-GTP binding to importin beta disrupts its binding to ligands- Ran-GDP does not bind Karyopherin/importin beta’s Role for Ran-GTP as a landmark for DNA- RanGEF Mitotic spindle assembly Nuclear envelope assembly Proof that Ran serves as the landmark for the assembly of the nuclear envelope around chromatinbeads beads membrane Sepharose beads loaded with Ran -GTP induce NE and nuclear pore complex assembly in Xenopus egg extracts. Immunofluorescence detection of Ran (left panels) and lipid staining by DHCC (right) of beads incubated in Xenopus egg extracts for 120 min. DAPI was used to stain Sepharose beads; no DNA was present. control inhibitor Nuclear envelope assembly Requires Ran at two steps: membrane fusion and NPC assembly Bidirectional traffic of macromolecules across the nuclear envelope- Thousands of proteins and ribonucleoproteins cross the nuclear envelope every second; mostly proteins involved in the synthesis of DNA, RNA and ribosomes. Nuclear Import ribosomal proteins transcription factors replication factors nucleolar proteins snRNA rRNA viral genomes Nuclear Export ribosome subunits mRNA tRNA snRNA rRNA viral RNP’s The nuclear pore complex functions as a permeability barrier Simple Diffusion NES - Facilitated Transport via Targeting Signals -NLS > 30 kDa Nuclear envelope Chromatin Tested experimentally using fluorescent dextrans of known sizes. NES - -NLS < 3 nm < 30 kDa Tested experimentally using fluorescent protein of large sizes or even gold particles with attached NLSs no protein unfolding is required; the signals are not cleaved Identification of the first nuclear localization signal- Large T-antigen NLS + + + ++ Pro Lys Lys Lys Arg Lys Val Karyopherins recognize NLS or NES containing proteins and “carry” them across the NPC - Yeast Karyopherins Karyopherin/importin β Karyopherin/ importin α Importins Kap95p-Kap60p Kap104p Kap121p Kap123p Mtr10p Nmd5p Sxm1p Lph2p Pdr6p Kap114p Ntf2 Exportins Crm1p Kap120p Los1p Cse1p Mex67 Transportins Msn5 Different types of nuclear localization and nuclear export signals- •  A typical NLS is composed of 5-7 basic amino acids clustered in one domain, or in two domains separated by a“spacer’ of 10 amino acids. A typical NES is composed of a stretch of leucines in a compact domain. NLS’s and NES’s are not cleaved off after translocation; they are used many times, usually after each cell cycle Regardless of size, some small nuclear proteins contain an NLS to promote efficient accumulation in the nucleus. •  •  •  RanGTP controls the directionality of protein transport by controlling the loading and unloading of transport cargos on karyopherins NES NES exportin Facilitated Transport via Karyopherins -NLS importin The Ran-GTP gradient Ran GDP GAP GDP GTP GDP GDP Ran GDP GAP Nuclear envelope Chromatin NES exportin -NLS GEF GDP GTP GTP GTP GTP GTP GTP Ran GTP -NLS importin GTP Ran Regulation of protein import and exportUsually exerted at the level of “availability” of the substrate’s NLS or NES Ligand-induced Conformational change Masking Anchoring NFAT’s ‘merry-go-round’ across the NPC ...
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This note was uploaded on 01/17/2011 for the course BIOL 110 taught by Professor Rexach during the Fall '10 term at UCSC.

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