-1- The Nucleus, Cell Cycle and Cell Division

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-1- The Nucleus, Cell Cycle and Cell Division Powered By Docstoc
					                         The Nucleus, Cell Cycle and Cell Division

CTS/Physiology Lecture #11         September 14, 2004             Dr. Robert A. Bloodgood

Learning Objectives:

•     What are the functions of the nucleus?
•     What is the structural organization of the nucleus
•     What is chromatin and how is it organized?
•     What are the stages of the mammalian cell cycle?
•     How is the cell cycle regulated?
•     What is the mechanism of mitosis and cytokinesis?
•     How does meiosis differ from mitosis?

I.    The Nucleus

A.    Living cells can be separated into prokaryotes, those without a nucleus, and eukaryotes,
      those with a nucleus. Evolution of a nucleus had a number of consequences.

      1.     Spatial and temporal segregation of transcription (occurring in the nucleus) from
             translation (occurring in the cytoplasm).
      2.     Development of extensive processing of RNAs.
      3.     A mechanism was needed for the transport of mRNAs, tRNAs and ribosomal subunits
             from the nucleus (site of synthesis, processing and assembly) to the cytoplasm (site
             where they function in protein synthesis).
      4.     Transcriptional control became more sophisticated with transcription factors
             synthesized in the cytoplasm, imported into the nucleus and regulated by
             phosphorylation.
      5.     A highly structured nucleus and a highly organized machine for gene
             segregation (mitotic apparatus) needed to handle the larger amounts of
             DNA found within eukaryotic cells and the demands of import and export.

B.    Functions of the Nucleus

      1.     Storage of information (in DNA)
      2.     Replication of the genome (DNA replication)
      3.     DNA repair
      4.     RNA synthesis (transcription)
      5.     RNA processing
      6.     Ribosome assembly
      7.     Transport of tRNAs, mRNAs and ribosomal subunits to the cytoplasm
      8.     Selective protein uptake from the cytoplasm

      There is localization of function in the nucleus. For instance, different classes of RNA are
      synthesized by different classes of RNA polymerase in different locations within the nucleus.


      Information flow occurs in both directions between the nuclear and cytoplasmic
      compartments. Information stored in the nucleus regulates the activities of the cytoplasm
      (DNA to mRNA to protein). Information from outside the cell sets up cytoplasmic signaling
      cascades which result in activation of transcription factors which regulate gene expression in
      the nucleus.


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C.   Overview of Nuclear Structure. The nucleus is generally the largest organelle in the cell and
     is bounded by two membranes. Major structures of the nucleus are the chromatin, nucleolus,
     nuclear envelope and nuclear matrix (nucleoplasm).

D.   Nuclear number and nuclear shape can vary in different cell types. Most cells have one,
     centrally located nucleus. There are a number of exceptions to a single nucleus.

     1.     Liver hepatocytes have a variable number of nuclei (1-3).
     2.     Osteoclasts and striated muscle fibers are multinucleated.
     3.     Lens fibers and erythrocytes have lost their nuclei.
     4.     Most nuclei are round or oval.
     5.     Certain nuclei are very irregular in shape. Good examples of this
            include the nuclei of mature sperm and neutrophils
            (polymorphonuclear leukocytes).
     6.     Changes in nuclear shape and appearance are associated with
            pathological conditions, including cancer.

E.   Chromatin

     1.     Chromatin is made up of DNA and protein.
     2.     The staining properties of chromatin reflect the functional state of
            the nucleus.
     3.     There are said to be two states of chromatin: heterochromatin and
            euchromatin.

            a.     Euchromatin is active in transcription (RNA synthesis).
            b.     Euchromatin stains poorly.
            c.     Heterochromatin is tightly packed and is not active in transcription
            d.     Heterochromatin stains heavily with basic dyes; it is basophilic.
            e.     The relative amounts of heterochromatin and euchromatin reflect the metabolic
                   activity of the cell.
            f.     Euchromatin and heterochromatin can interconvert.

     4.     The state of the chromatin can change with the stage of differentiation of a cell or with
            the stage of the cell cycle. Extreme chromatin condensation gives rise to
            chromosomes during M phase and can occur in certain post-mitotic cells (red blood cell
            precursors before ejection of the nucleus).

     5.    There is a hierarchy of DNA packing within chromatin.

            a.     The nucleosome (10-11 nm in diameter) is the basic unit of chromatin
                   organization
            b.     The nucleosome has a stretch of DNA wrapped around a protein core made up
                   of 2 copies each of histones H2A, H2B, H3, H4.
            c.     Histone H1 is located between nucleosomes. It promotes the helical packing of
                   nucleosomes to form the 30 nm chromatin filaments (referred to as the solenoid
                   structure).
            d.     Histone methylation can control the transition between euchromatin and
                   heterochromatin and can regulate transcription.
            e.     Higher order packing of chromatin occurs during chromosome
                   condensation in prophase of mitosis.



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F.    Structure and Function of the Nucleolus

       1.      The nucleolus is spherical in shape; most (but not all) cells have a single nucleolus.
       2.      The nucleolus stains with basic dyes because of its content of DNA and RNA.
       3.      Nucleoli are sometimes surrounded by heterochromatin that is referred to as the
               nucleolus-associated DNA.
       4.      The nucleolus is the site of synthesis and processing of the 45S rRNA precursor.
       5.      The nucleolus is the site of assembly of the ribosomal large and small subunits.
       6.      Nucleoli are organized around the nucleolar organizer DNA (the DNA coding for the
               many copies of the 45S rRNA precursor).

G.     Nuclear Envelope

       1.      The nuclear envelope defines the nuclear compartment and consists of two
               membranes enclosing a cisternal space.
       2.      A nuclear lamina composed of three proteins (lamins A,B,C) is associated with the
               inner nuclear membrane. Specific regions of chromatin are anchored on the nuclear
               lamina. The phosphorylation state of the lamins regulates the breakdown and
               assembly of the nuclear envelope during the M phase of the cell cycle.
       3.      The nuclear envelope contains many nuclear pores which regulate all transfer of
               materials between the nucleus and the cytoplasm. mRNAs, tRNAs and ribosomal
               subunits leave the nucleus. A variety of proteins (histones, ribosomal proteins, RNA
               and DNA polymerases and transcription factors) enter the nucleus.
       4.      The outer nuclear membrane binds ribosomes and functions like rough endoplasmic
               reticulum.

II     The Cell Division Cycle

        Somatic cells reproduce by a highly regulated process called the Cell Division Cycle. Cell
multiplication is needed during embryogenesis, for normal turnover of cells in adult tissues, for
immune responses and for wound repair. The rate of cell turnover differs dramatically between
different tissues of the human body. There is a high rate of turnover in epithelial cells in the skin and
gut and in the hematopoietic cells. On the other hand, there is little or no turnover of neurons and
skeletal muscle cells. Defects in the regulation of cell division result in cancer.

A.    Steps in the cell cycle

       1.     The normal cell cycle is said to have four phases, G1, S, G2, M.
       2.     Collectively, G1, S and G2 are called interphase.
       3.     Human somatic cells are said to be diploid. They have the diploid number of
              chromosomes (46) throughout interphase. They have the diploid amount of DNA
              during G1.
       4.     Cells that are terminally differentiated are often removed from the cell cycle. They are
              said to be in the Go phase of the cell cycle, a special case of G1.
       5.     DNA synthesis occurs in the S phase
       6.     Mitosis and cytokinesis occur in the M phase

B.    Regulation of the cell cycle

       1.      The cell cycle must be very carefully regulated
       2.      There is a certain commitment step that occurs in G1. Once that has been passed, the
               cell must go through the cell cycle or undergo apoptosis (programmed cell death)


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     3.     Certain conditions must be fulfilled in each step of the cell cycle before the cell can
            progress to the next. These are monitored by cell cycle checkpoints.
     4.     The cell cycle is regulated by protein kinases, protein phosphatases, proteases and
            cyclins (proteins whose amount varies through the cell cycle).
     5.     There are two primary points at which the cell cycle is regulated: the G1 to S transition
            and the G2 to M transition
     6.     The major regulators of the cell cycle are Cyclin Dependent Kinases (CDKs)
     7.     Defects in regulation lead to cancer. Oncoproteins (coded by oncogenes) cause
            inappropriate cell growth by subverting growth factor pathways. Tumor suppressor
            genes (such as the retinoblastoma gene) normally suppress cell growth; defects in
            these genes can lead to uncontrolled cell growth.

C.   Cyclin-dependent kinases (CDKs)

     1.     CDKs regulate the major steps in the cell cycle by phosphorylating a variety of cellular
            proteins. Different CDKs and different cyclins function at different stages in the cell
            cycle.
     2.     CDKs are phosphoproteins that are regulated by phosphorylation and by the binding of
            cyclin proteins.
     3.     Cyclins accumulate in amount
            during specific phases of the cell
            cycle and then are destroyed by
            proteolysis.
     4.     CDKs are regulated by both
            activating and inhibitory
            phosphorylations.
     5.     M-phase promoting factor (MPF)
            regulates entry into mitosis and
            consists of CDK1 (also called
            CDC2) and cyclin B.

D.   Cell cycle checkpoints (three major types of checkpoints)

     1.     A checkpoint insures that all DNA damage has been corrected prior to entry into S
            phase
     2.     A checkpoint insures that all DNA has been duplicated before the cell enters mitosis
     3.     A spindle checkpoint ensures that all chromosomes have been aligned on the
            metaphase plate before anaphase is allowed to begin
     4.     The cell cycle checkpoints can result in a cell entering apoptosis (for instance, if there
            is too much DNA damage to be repaired). Apoptosis is programmed cell death that
            can result from signals external or internal to the cell. In either case, proteolytic
            enzymes called caspases are activated. In many cases, apoptosis is activated by
            release of cytochrome C from mitochondria.
     5.     Defects in cell cycle checkpoints can lead to a variety of pathological conditions,
            including cancer and genetic defects.

E.   Cancer therapy targets components of the cell cycle. Most cancer chemotherapeutic drugs
     target dividing cells (and are not specific for cancer cells). Specific targets are DNA synthesis
     (S phase) and mitosis (M phase). Drugs like Methotrexate (5-fluorouracil; a nucleoside
     analog) and Topoisomerase II inhibitors interferes with DNA synthesis (S phase). Drugs like
     vinblastine and Taxol interfere with microtubule dynamics and mitosis. DNA damage induced
     by radiation and chemotherapy can induce apoptosis.


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F.     M phase of the cell cycle involves two very different mechanisms. Mitosis results in
duplication of the nucleus followed by cytokinesis which results in duplication of the cells. A special
case of cell reproduction involves production of gametes and is called gametogenesis and involves
the process of meiosis instead of mitosis. Mitosis and meiosis utilize an elegant microtubule-based
machine called the spindle. Mitosis and meiosis involve both microtubule based motor proteins and
microtubule assembly and disassembly. On the other hand, cytokinesis involves actin machinery and
myosin motor proteins.

       Mitosis

       1.      The goal of mitosis is to bring about gene segregation. In S phase, all the
               chromosomes are duplicated giving two chromatids per chromosome; during mitosis,
               each of the 46 chromosomes is split in half and one chromatid goes to each daughter
               cell.

       2.      Structure of the spindle: The two spindle poles are centrosomes, structures that
               nucleate all of the microtubules of the spindle. Functionally, there are three types of
               spindle microtubules: 1) aster microtubules, 2) kinetochore microtubules (attached to
               chromosomes) and 3) interpolar microtubules.

       3.      The stages of mitosis in animal cells are:

               Prophase: Chromosomes condense inside the nucleus; each chromosome has two
               daughter chromatids and a structure called the kinetochore, which will be the point of
               attachment to the spindle. Outside the nucleus, the basal bodies separate, form the
               spindle poles and nucleate microtubules that will organize into the spindle.

               Prometaphase: This stage begins at the time of nuclear envelope breakdown, which
               allows the condensed chromosomes to interact with the spindle microtubules. Certain
               microtubules capture chromosomes. Microtubule assembly and motor proteins move
               the chromosomes to the middle of the spindle in a process called congression.

               Metaphase: This stage is achieved when all of the chromosomes are properly aligned
               at the middle of the spindle forming a metaphase plate. A spindle checkpoint operates,
               which delays the onset of anaphase until all of the chromosomes are aligned.

               Anaphase: This is the stage in which proteolytic events separate the pairs of
               chromatids and a combination of microtubule depolymerization, microtubule
               polymerization and motor proteins move the daughter chromatids to opposite spindle
               poles. Anaphase consists of two parts: anaphase A and anaphase B.

                      Anaphase A: Movement of the chromatids to the spindle poles by the
                      combined action of a motor protein (dynein) located at the kinetochore and
                      kinetochore microtubule depolymerization.

                      Anaphase B: Separation of the spindle poles (elongation of the spindle) is due
                      to the assembly of the interpolar microtubules and the sliding of the interpolar
                      microtubules due to the action of a motor protein (kinesin-like proteins). May
                      also involve a motor protein (dynein) pulling on the aster microtubules.

               Telophase: During this stage, the daughter chromatids stop moving, decondense and
               the nuclear envelope reforms.


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       Cytokinesis is the process by which the cell produces two daughter cells. It usually begins
       prior to the completion of anaphase and involves a localized array of actin filaments attached
       to the plasma membrane. Myosin induces actin filament sliding and pinching in of the plasma
       membrane.

G.    Meiosis is the production of germ cells containing half the number of chromosomes
      and half the amount of DNA found in normal somatic (Go or G1) cells

       1.     Two rounds of spindle-based activity occur (Meiosis I and Meiosis II) without an
              intervening S phase.

       2.     During prophase of meiosis I homologous chromosomes pair together. During this
              pairing pieces of DNA are exchanged between homologous chromosomes in a
              process called crossing over. Crossing over plus the independent assortment of
              maternal and paternal chromosomes contribute to heterogeneity of the genetic material
              in the gametes.

       3.     During anaphase of meiosis I the
              homologous chromosomes move apart.
              The chromosome number in human is thus
              reduced from 46 to 23 but each of these
              chromosomes still contains two chromatids.
              The amount of DNA is reduced in half.

       4.     During Meiosis II each chromosome splits
              into two chromatids which are moved to
              opposite poles. The amount of DNA is
              again reduced in half. In many ways,
              meiosis II resembles mitosis except that
              only 23 chromosomes are involved instead
              of 46.

       5.     The outcome of meiosis is that four cells
              are produced from the original G2 cell; each
              has half the normal number of
              chromosomes (23) and half the normal G1
              amount of DNA (haploid). In males, all four
              products develop into sperm; in females,
              only one of the four becomes an egg.             Comparison of mitosis and meiosis

       Malfunctions in meiotic machinery can result in cells that have too few or too many
       chromosomes and are termed aneuploid. Most human aneuploidy results from abnormal
       segregation of chromosomes during meiosis I (non-disjunction). This can result from a
       malfunction of the spindle check-point. An example is Down’s Syndrome, where the embryo
       has three copies of chromosome 21 (2 from one gamete and 1 from the other gamete).

Reading:

Gartner and Hiatt, 2nd ed, pp 51-70; Young and Heath (Wheaters Atlas), 4th edition, pp. 33-44.
Supplementary resource: Alberts et al. Molecular Biology of the Cell, 4th Ed., Chapters 17 & 18.




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