nucleus by panniuniu

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									Cell nucleus
           Nucleus

 Repository for genetic material
 Directs activities of the cell
 Usually single, some cells several, RBC none
   Nucleolus - region of intensive ribosomal RNA synthesis

 Surface of nucleus bound by two phospholipid
  bilayer membranes
      nuclear membrane
      Nuclear pores – protein gatekeepers
         Usually   proteins going in and RNA going out
Nucleus
Cell Division: Nomenclature

 Only for eukaryotes:
 mitosis normal cell division of somatic cells;
  from diploid -> 2 x diploid chromosome set
 meiosis generation of germ cells (reduction
  division); from diploid -> 4x haploid
  chromosome set
For prokaryotes often no differentiated phases
  between metabolism and DNA-replication, but
  all phases simultaneously.
Haploid or diploid

 Diploide cells have a doubled chromosome set
 (i.e. human: 2 x 23).
 Haploide cells have a single chromosome set
 (i.e. most fungae and algae). Also human
 sperm and egg cells (gametes) are haploid.
 Polyploid cells contain many chromosome sets
 (many agricultural plants, by cultivation).
 -> larger nucleus -> larger cell -> larger fruit
               Human Chromosomes
 a) chromosome set:
 Arrangement and
    enumeration of autosomes
    (non-allosome) with
    decreasing size. In the body
    cells the chromosomes 1-22
    are doubled
   X, Y: allosomes determining
    gender.
   b) Structure of a
    chromosome from wound
    strands of desoxyribonucleic
    acid (DNA); P = phosphoric
    acid, Z = sugar,
   C = cytosine, G = guanine, T
    = thymine, A = adenine
   2 chromatides per
    chromosome
   Each chromatin strand
    consists from double helix
   human: 2 x 22 + 2 of DNA
                      Three DNA sequences
                      required to produce a
                      Chromosome:



1. 2 Telomeres
(replication and
prevent sticking)
2. Centromere
(Kinetochore)
(attaching daughter
chromosomes)
3. 2 replication
origins
Allele
 One gene to be inherited,
    exists
   in two copies (alleles)
   Both gametes contain
    only one
   copy.
   Y is dominant, the pea is
    yellow
   genotype Yy = phenotype
    yellow
   (Gregor Mendel, 1865)
Genotype and Phenotype

 Genotype is the specific set of alleles of
  an individuum, the construction plan.
 Phenotype is the realized appearance of
  an individuum, the end product.
Chromosome-Structures
Nucleosome
             In chromosome the
             double stranded DNA
             is wrapped into proteins
             (Histones).
             This wrapping body is
             called nucleosome and
             each contains 2 Histones
             H2A, H2B, H3 und H4,
             and the linker-histone
             H1. The DNA is wrapped
             with 2 windings.
Solenoide




 For further wrapping all H1 Histones lie together
and form an further wrapping body, the solenoide
(chromatosome) with approx. 30 nm diameter.
     Chromosome




The solenoides form straps around the
scaffold-proteine: 300 nm structures
        Structure of DNA

 Double strand consists of
-spine (here dark)
(sugar - phosphate)
and
-ladder steps (here red)
(base pairs)
 0,34 nm step distance
 3,4 nm periodicity
 2 nm diameter
                                   31
Visualizing transcription (rRNA)

RNA polymerase
                                            32
Nucleolus is a ribosome-producing factory
             33




           txn
assembly
                               34
Nucleoli dissociate and fuse
Fibrillarin (sno RNP)   Pre-mRNA splicing                      35


                                            Subnuclear
                                            structures:

                                            Nucleoli,

                                            Cajal bodies,
                                            GEMS,

                                            Interchromatin
                                            Granule clusters
                                            Or speckles,


  Chromatin
                 Mitosis
To remember:
„Pro-Me-An-Te“
       Meiosis
       (Germ cells)
 From a cell with
  diploid set to four
  cells with haploid
  chromosome set
 Each of the double
  chromosomes is
  separated into its
  same equal
  chromatides.
 => 4 times same
  information
                  Comparison Mitosis - Meiosis
   First: DNADuplication
   and then
   Condensation




                                          In each case:
                                          Separation of
                                          chromatides
                                          and
                                          division on
                                          daughter cells
 The Cell Nucleus


Structure,Trafficking, and Organized
Function


 References:
 Molecular Biology of the Cell. Alberts, 4th edition
 Chapt. 12 pages 669-678
 Chapt. 6 pages 331-335
 Molecular Cell Biology. Lodish, 4th edition
 Chapt. 11 pages 426-435.
Nuclear Structure,Trafficking, and Organized
Function

 Introduction
 Detailed nuclear structure and functional organization
-cross-section of a typical nucleus
-nuclear envelope
-nuclear lamina
-nucleolus structure and function
-nuclear pore structure and function
 Nuclear trafficking signals and mechanisms
 Diseases associated with alterations to nuclear
  proteins
cross-section of a typical nucleus
                                             Nuclear pores



                                           Nuclear Pores




Lodish 5-50: freeze fracture preparation
Nuclear Pore Complexes (NPC)
  NPCs span the inner and outer nuclear membrane
     3000-4000 in typical mammalian cell nucleus.

  NPC are:
     large - 125 million Da
     complex- composed of more than 50 different proteins
     gated - Have diffusion limit of 50-60 kDa. Larger proteins
      require active transport
     busy - every minute each NPC must transport 100 histone
      proteins, 6 small and large ribosome subunits, plus
      numerous other proteins and RNP complexes.
     traffic is bi-directional and highly regulated.
Transport through NPCs
 Import (chromatin proteins, ribosomal proteins RNA
  processing proteins, proteins that shuttle between
  nucleus and cytoplasm)
      players:
         nuclear import receptors
         The GTPase Ran and its regulators
         Sequence on protein to be imported -Nuclear localization
          signal (NLS)
 Export (ribosome subunits, mRNA complexes, tRNAs,
  shuttling proteins )
      players:
         nuclear export receptors
         Ran and its regulators
         Sequence on protein to be exported - Nuclear export
          signal (NES)
Nuclear pores- complex structures with 8-fold
                 symmetry
Two mechanisms for
nuclear/cytoplasmic transport
 small molecular weight molecules can pass by
  diffusion (energy independent)
      < 5000 daltons - freely permeable
      ~17,000 daltons - 2 min equilibration
      > 60,000 daltons- impermeable to nuclear entry.
 But what about ribosomal subunits, mRNP
  particles with molecular weights in millions?
Large molecules must be actively
transported through the nuclear pore (energy
dependent)

 How do investigate this process?
      identify how proteins are targeted for nuclear
       import/export.
      identify proteins and determine the structure of nuclear
       pore.
      reconstitute transport process in vitro.
Nuclear trafficking signals

 nuclear localization sequence (NLS)
    often basic amino acid sequence
    single sequence or two separate sequences
     (bipartite NLS)
 nuclear export sequence (NES)
    often leucine rich


       Example 1: LVRELEDIRL

       Example 2: LKLLTTADLQIL
 Proteins required for nuclear import


  Nuclear transport receptors

               RanGTP-binding               Importin a binding
Importin b

  Nuclear transport adaptors
             IBB        Basic NLS-binding     IBB = importinb binding
Importin a                                    domain




  Ran: small GTPase            GTP              GDP
Active nuclear transport is driven
by Ran
 Ran is a small GTPase- binds and hydrolyses guanine
  nucleotide triphosphate (GTP)
      Ran exists in two conformational forms: Ran - GTP and
       Ran-GDP
      Ran has weak GTPase activity that is stimulated by
       RanGAP (GTPase activating protein)
      A guanine nucleotide exchange factor (GEF), called
       RCC1 stimulates the release of GDP and binding of
       GTP (higher concentration of GTP in the cell assures
       preferential binding of GTP)
      The cycle of GTP binding, hydrolysis, release provides
       energy for nuclear transport
      asymmetric localization of RanGAP and RCC1 assure
       directionality of transport
Ran- prime mover in nuclear transport
Not all proteins that enter the nucleus
have NLS sequences
 Ran and its cofactors RanGAP, and RCC1 are required
 for additional aspects of nuclear function: nuclear
 membrane assembly, nuclear pore assembly, spindle
 assembly




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   nuclear membrane                                    nuclear membrane in
                                                       the absence of Ran
Peter Askjaer, Vincent Galy, Eva Hannak, *and Iain W. Mattaj. Mol
Biol Cell. 2002 December; 13 (12): 4355–4370
Nuclear import/export is key
regulatory step


      Many examples:
      dorsal




      Control of T-cell activation
        by nuclear import
cyclosporin A

   FK506
 Objectives
1. The nucleus is highly organized
    Know the structure and function of the nucleus and the nuclear
   envelope.
    Understand the function of the nucleolus and how it relates
   to structure.

2. Nuclear import and export are receptor-mediated events
    Know how nuclear import occurs
    Know how nuclear export occurs for NES-containing proteins
    Know regulatory mechanisms controlling nuclear import of proteins

3. Nuclear lamins are targets of mutations that lead to disease
    Be able to discuss Emery-Dreifuss muscular dystrophy (EDMD)
    Be able to discuss Dunnigan-type familial partial lipodystrophy

								
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