The centrosome in cell cycle and signal transduction pathways by steepslope9876



     The centrosome in cell cycle and
     signal transduction pathways
     Bodo M.H. Lange
     Classically the centrosome is known as        they provide a dominant site for micro-              the elucidation of the centrosome com-
     the microtubule organising centre in          tubule nucleation and play a role in                 position (Andersen et al. 2003; Bornens
     higher eukaryotic cells, coordinating         positioning the spindle correctly in the             2002; Doxsey 2002; Wigge et al. 1998)
     microtubule-dependent functions. Alt-         cell (Rieder et al. 2001).                           however, we still don’t know the majori-
     hough correct, this is only part of the       Why does this cell organelle respond to              ty of components of the centrosome. If
     story of what a centrosome does. The          global cellular signalling cues after all?           we really want to untangle the molecu-
     multitude of the microtubule-dependent        One answer might be that it is part of a             lar spider web of signal transduction, cell
     functions in a cell (including intracellu-    complex cellular protection mechanism                cycle regulation and centrosome func-
     lar transport, cell shape and polarity        that brings the cell cycle to a stop when            tion then, no doubt, we have to tackle
     determination, motility, mitosis, cytoki-     environmental conditions are harmful.                this problem. Luckily, we have now new
     nesis) demands that the microtubule           At the moment we don’t know how such                 technologies and knowledge at our
     organising centre is both regulated by        signal transduction pathways are inter-              hands and the next section will give an
     and regulating cell cycle and signal          connected to the centrosome and which                overview on how we are addressing this
     transduction events. In this sense the        centrosomal proteins are substrates for              problem in our laboratory.
     centrosome is "more" than a microtubu-        signal transducer molecules. For sure,
     le-organising centre, playing an active       some of the answers to these questions               A systematic screen of centrosome
     role also in cell cycle progression, stress   lay in the molecular composition and                 composition and function
     response and checkpoint control, via          organisation of the centrosome and thus              In our work we are focusing on three
     interactions with numerous signal trans-      reflecting its multiple functions. In this           fundamental questions: (1) How is the
     duction molecules (Lange 2002). For           respect, good progress has been made in              centrosome integrated in cell cycle and
     example damaged or incompletely repli-
     cated DNA triggers centrosome destruc-
     tion in Drosophila early embryos (Takada
     et al. 2003) suggesting an interconnec-
     tion of the centrosome to DNA damage
     signalling pathways. In addition, follo-
     wing destruction of the centrosome in
     cultured cells by laser beam irradiation,
     a mitotic spindle is formed, chromoso-
     mes are separating, but the cells under-
     go abnormal cytokinesis and fail to exit
     the G1 phase of the cell cycle (Khodjakov
     & Rieder, 2001). This data suggests that
     the centrosome has a primary role in
     cytokinesis and cell cycle progression
     rather than in spindle formation. Indeed,
     there is not an absolute requirement for
     cells to possess a centrosome to form
     spindles. Spindles can be formed
     without centrosomes in naturally occur-
     ring systems such as in Drosophila oocy-
     tes (Theurkauf & Hawley, 1992) or in
     vitro systems (Heald et al. 1996) where
     motor proteins rather than centrosomes
     have been proposed to mediate spindle
     formation. This does not mean, however,
                                                   Fig. 1: Immunofluoresence microscopy image of the preblastoderm stage of a Drosophila
     that centrosomes are superfluous for          embryo labeled with an antibody against phosphorylated histone H3. The condensing
     spindle and microtubule organisation,         prophase chromosomes, shown in red, demonstrate the synchronous duplication and
     because when centrosomes are present          division cycles in the early embryo syncytium.

18 Zellbiologie aktuell · 30. Jahrgang · Ausgabe 1/2004

                                                      In order to answer these questions we          cost effective method to knock down
                                                      first need to have more complete infor-        protein expression from a gene of known
                                                      mation of the molecular make-up of the         sequence (Figure 3). The obtained phe-
                                                      centrosome. The centrosome has so far          notypes are classified according to their
                                                      escaped detailed biochemical and func-         relevance for centrosome function and
                                                      tional characterisation due to the small       integration in signal transduction path-
                                                      quantities of protein obtained through         ways. Subsequently, relevant proteins
                                                      organelle isolation methods and due to         are cloned, their localisation is verified
                                                      lack of genetic approaches to study its        through GFP-tagged expression and
                                                      function in higher eukaryotes. However,        characterised in flies. Furthermore, with
                                                      new and improved methods, such as bet-         the biochemical characterisation of the-
                                                      ter isolation techniques (Lange et al.         se proteins we identify specific protein-
                                                      2000a; Müller, Lehmann & Lange unpu-           protein interactions between novel or
                                                      blished), improved sensitivity of mass         known centrosomal and signalling mole-
                                                      spectrometry (Yarmush & Jayaraman,             cules. In summary, we developed a very
                                                      2002) and RNA interference (Clemens et         efficient functional screen that integra-
                                                      al. 2000) are making a systematic bio-
                                                      chemical and functional dissection of
                                                      this organelle feasible.
                                                      To address these questions we initiated a
                                                      systematic analysis of the molecular
                                                      components of the centrosome using an
                                                      affinity-purification isolation method in
                                                      combination with mass spectrometry
                                                      protein identification. We are using
                                                      Drosophila as a model system for this
                                                      work. Why Drosophila? First, Drosophila
                                                      is an organism that is genetically mani-
                                                      pulable and its genome has been
                                                      sequenced. Therefore, we can study the
                                                      function of individual molecules in the
                                                      context of tissue and throughout deve-
                                                      lopment, providing functional clues that
                                                      would be absent in a cell culture system.
                                                      Secondly, the syncytial (cell membrane
                                                      free) early embryonic stage of Drosophi-
Fig. 2: (A, B) Immunofluoresence microscopy
images of isolated Drososphila centrosomes
                                                      la is well suited for the isolation of cen-
labelled with an anti- -tubulin antibody.             trosomes (Figure 1): these embryos are
(A) Centrosomes isolated by sucrose density           essentially tiny "bags”, not constrained
centrifugation. (B) Centrosomes further purified      by cellular borders, full of cell organelles
by immuno-affinity isolation on magnetic beads        and native protein complexes that are
coupled to antibodies. Beads are shown in red,
centrosomes in yellow. (C) MALDI mass spectro-
                                                      easily amenable to biochemical isola-
metry fingerprinting of the centrosomal prepara-      tion. Thirdly, growing large fly popula-
tions isolated from coomassie-stained gels. The       tions we can obtain per day 30-50 g of
most abundant band identified (green circle) is the   embryo starting material, required for
protein CNN. The diagram shows the hits of the        the systematic mass isolation and cha-
peptide mass distribution that was used for the
identification of the CNN protein, after tryptic
                                                      racterisation of centrosomes.
digest (work in collaboration with Gustavsson         To isolate centrosomes we developed an
& Gobom).                                             immuno-affinity approach using magne-
                                                      tic beads (Figure 2). This method has the
                                                                                                     Fig. 3: Schematic flow diagram of the RNAi
                                                      advantage that most contaminants               functional screen: Once centrosomal proteins
cell signalling pathways? (2) What holds              (which otherwise co-migrate with cen-          have been identified by MALDI mass spectrometry,
the structure of the centrosome together              trosomes in biochemical fractionation          dsRNA is generated by PCR amplification from
and constitutes the scaffold for the                  methods) are removed. In this way we           genomic target DNA sequences. dsRNA is used for
assembly of functional protein comple-                                                               the protein “knock-down” assay in cultured Droso-
                                                      have identified over 60 centrosomal pro-
                                                                                                     phila cells. Phenotypes are analysed by immuno-
xes onto the centrosome? (3) Which                    tein candidates. We are screening those        fluorescence microscopy: centrosomes are labelled
molecular events mediate the change of                candidate molecules for their relevance        with anti- -tubulin antibodies (green), DNA is
microtubule nucleation capacity of the                by RNAi in Drosophila cultured cells.          stained with DAPI (blue) and microtubules with
centrosome during the cell cycle?                     This is a fast, extremely efficient and        anti- -tubulin antibody (red).

                                                                                           Zellbiologie aktuell · 30. Jahrgang · Ausgabe 1/2004          19

     tes the direct biochemical identification     get kinases to Hsp90, it was likely that               los, P. Becker, A. Hyman and E. Karsenti. 1996. Self-
     of novel centrosomal proteins with their      the observed phenotypes could be                       organization of microtubules into bipolar spindles
                                                                                                          around artificial chromosomes in Xenopus egg
     functional characterisation. The follo-       brought about by the inactivation of one               extracts. Nature 382, 420-425.
     wing sections describe two examples of        or more kinases. Indeed, the inactivation              Khodjakov, A. and C.L. Rieder. 2001. Centrosomes
     the application of this approach.             of Hsp90 and Cdc37 lead to degradation                 enhance the fidelity of cytokinesis in vertebrates
                                                   of Aurora B kinase in mammalian cells                  and are required for cell cycle progression. J. Cell
     Centrosome function depends on                and in Drosophila (Lange et al. 2002).                 Biol. 153, 237-242.
     Hsp90                                         Hence, the requirement of the                          Lange, B.M.H. 2002. Integration of the centrosome
     One of the centrosomal proteins identi-       Cdc37/Hsp90 complex for mitotic pro-                   in cell cycle control, stress response and signal
     fied, heat shock protein 90 (Hsp90) is a                                                             transduction pathways. Curr. Opin. Cell Biol. 14,
                                                   cesses and microtubule organisation is                 35-43.
     molecular chaperon maintaining the            evolutionary conserved.
                                                                                                          Lange, B.M.H., E. Rebollo, A. Herold, and C. Gonza-
     stability and activity of signalling mole-
                                                                                                          lez. 2002. Cdc37 is essential for chromosome
     cules (Lange et al. 2000a). Hsp90 is pre-     Outlook                                                segregation and cytokinesis in higher eukaryotes.
     sent in the Drosophila centrosome in dif-     Our approach integrates the direct bio-                EMBO J., 21, 5364-5374.
     ferent developmental and cell cycle sta-      chemical identification of novel centro-               Lange, B.M.H., A. Bachi, M. Wilm and C. Gonzalez.
     ges, in the early embryo as well as in lar-   somal proteins with their functional                   2000a. Hsp90 is a core centrosomal component
     val spermatocytes. Moreover, Hsp90                                                                   and is required at different stages of the centroso-
                                                   characterisation. This combination suc-
                                                                                                          me cycle in Drosophila and vertebrates. EMBO J.
     localisation at the centrosome is conser-     cessfully exploits the advantages of the               19, 1252-1262.
     ved in all vertebrate cells from chicken      excellent biochemical and genetic sys-                 Lange, B.M.H., A.J. Faragher, P. March and K. Gull.
     to human. We demonstrated that Hsp90          tem of Drosophila. So far, we have iden-               2000b. Centriole duplication and maturation in
     is a core centrosomal protein necessary       tified over 60 candidate centrosomal                   animal cells. Current Topics Dev. Biol. 49, 235-249.
     for the maintenance of centrosome             proteins which are currently being cha-                Rieder, C.L., S. Faruki, and A. Khodjakov. 2001. The
     function and integrity, required for the      racterised. With this system at hand, the              centrosome in vertebrates: more than a microtu-
     fidelity of chromosome segregation and        combination of cell biology approaches                 bule-organizing center. Trends Cell Biol. 11:413-
     cell cycle progression (Lange et al.          with the expertise and facilities for high-
     2000a). Abrogation of Hsp90 function in                                                              Takada, S, A. Kelkar and W.E. Theurkauf. 2003. Dro-
                                                   throughput analysis at the Max-Planck                  sophila checkpoint kinase 2 couples centrosome
     Drosophila or in mammalian cells results      Institute for Molecular Genetics in Berlin             function and spindle assembly to genomic integri-
     in abnormal centrosomal and mitotic           provides the opportunity to obtain a                   ty. Cell 11, 87-99.
     phenotypes. In particular, centrosomal        genome wide view of protein complex                    Theurkauf, W.E. and R.S. Hawley. 1992. Meiotic
     separation and maturation (i.e. the           structure and in vivo function. In addi-               spindle assembly in Drosophila females: Behavior
     increase in pericentriolar material during    tion, the structural characterisation of               of nonexchange chromosomes and the effects of
                                                                                                          mutations in the nod kinesin-like protein. J. Cell
     the cell cycle; Lange et al. 2000b) are       centrosomal protein complexes is ongo-                 Biol. 116, 1167–1180.
     affected, resulting in cells with fragmen-    ing, as part of the Ultra-Structural Net-
                                                                                                          Wigge, P.A., O.N. Jensen, S. Holmes, S. Soues, M.
     ted and abnormal in size centrosomes.         work (USN) in Berlin/Berlin-Branden-                   Mann and J.V. Kilmartin. 1998. Analysis of the Sac-
     Furthermore, lack of Hsp90 function           burg, which is aiming at the high-                     charomyces spindle pole by matrix-assisted laser
     triggers abnormal spindle formation           throughput isolation and structural ana-               desorption/ionization (MALDI) mass spectrometry.
     with the subsequent chromosome segre-                                                                J. Cell Biol. 141, 967-977.
                                                   lysis of protein complexes via cryo-elec-
     gation resulting in a high percentage of      tron microscopy. The elucidation of cen-               Yarmush, M.L. and A. Jayaraman. 2002. Advances
     aneuploid and polyploid cells. In sum-                                                               in proteomic technologies. Annu. Rev. Biomed. Eng.
                                                   trosome structure and function will help               4, 349-373.
     mary, Hsp90 has an essential role in cen-     us to understand the molecular events
     trosome function and mitosis in higher        and the regulation of cell division and
                                                                                                          Anschrift des Verfassers:
     eukaryotic cells.                             signal transduction in flies and humans.
                                                                                                          PD Dr. Bodo M. H. Lange
     Integration of microtubule organi-                                                                   Max-Planck Institute for Molecular Genetics
                                                   References                                             Department of Vertebrate Genomics
     sation in the cell cycle machinery            Andersen, J.S., C.J. Wilkinson, T. Mayor, P. Morten-   Ihnestrasse 73
     We extended our functional assay to           sen, E.A. Nigg, and M. Mann. 2003. Proteomic cha-      14195 Berlin
     Cdc37, the kinase-targeting unit of           racterization of the human centrosome by protein       Tel.: +49-30-8413-1645
     Hsp90 that is involved in the functional      correlation profiling. Nature, 426, 570-574.           FAX: +49-30-8413-1128
     maturation of protein kinases. Abroga-        Bornens M. 2002. Centrosome composition and            Email:
                                                   microtubule anchoring mechanisms. Curr. Opin.
     ting Cdc37 function (either by mutations
                                                   Cell Biol. 14, 25-34.
     or through “knock-down” RNAi experi-
                                                   Clemens J.C., C.A. Worby, N. Simonson-Leff, M.
     ments) in Drosophila demonstrated that        Muda, T. Maehama, B.A. Hemmings and J.E. Dixon.
     this Hsp90 co-factor is essential for mit-    2000. Use of double-stranded RNA interference in
     otic progression and cytokinesis (Lange       Drosophila cell lines to dissect signal transduction
     et al. 2002). Moreover, our experiments       pathways. Proc. Natl. Acad. Sci. U S A. 97, 6499-
     showed that Cdc37 is essential for chro-
     mosome condensation, chromosome               Doxsey, S. 2001. Re-evaluating centrosome func-
                                                   tion. Nat. Rev. Mol. Cell Biol. 2, 688-698.
     segregation and formation of a central
                                                   Heald, R., R. Tournebize, T. Blank, R. Sandaltzopou-
     spindle. Because Cdc37 is known to tar-

20 Zellbiologie aktuell · 30. Jahrgang · Ausgabe 1/2004

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