Hergert Polla

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  • Preprint
    Laser microsurgery in the GFP era : a cell biologist's perspective
    ( 2007-06) Magidson, Valentin ; Loncarek, Jadranka ; Hergert, Polla ; Rieder, Conly L. ; Khodjakov, Alexey
    Modern biology is based largely on a reductionistic ‘dissection’ approach – most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter we review some of the applications for laser microsurgery at the subcellular level, and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our lab. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope or software, but instead only in defining creative questions and in visualizing the target to be destroyed.
  • Preprint
    Control of daughter centriole formation by the pericentriolar material
    ( 2008-02) Loncarek, Jadranka ; Hergert, Polla ; Magidson, Valentin ; Khodjakov, Alexey
    Controlling the number of its centrioles is vital for the cell as supernumerary centrioles result in multipolar mitosis and genomic instability. Normally, just one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole ‘overduplication’ are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in Sarrested cells. Under conditions when multiple daughters simultaneously form on a single mother, all of these daughters must be removed to induce reduplication. Intriguingly, the number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) via overexpression of the PCM protein pericentrin in S-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.
  • Preprint
    Chromosomes can congress to the metaphase plate before biorientation
    ( 2005-12-09) Kapoor, Tarun M. ; Lampson, Michael A. ; Hergert, Polla ; Cameron, Lisa ; Cimini, Daniela ; Salmon, Edward D. ; McEwen, Bruce F. ; Khodjakov, Alexey
    The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle (i.e., “bi-oriented”). We found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy and correlative electron microscopy, we found that mono-oriented chromosomes could glide towards the spindle equator alongside kinetochore fibers attached to other already bi-oriented chromosomes. This congression mechanism depended on the kinetochore-associated plus enddirected microtubule motor CENP-E (kinesin-7).