Article

Influence of centriole number on mitotic spindle length and symmetry

Department of Biochemistry and Biophysics, UCSF, San Francisco, California 94158, USA.
Cytoskeleton (Impact Factor: 3.01). 08/2010; 67(8):504-18. DOI: 10.1002/cm.20462
Source: PubMed

ABSTRACT The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

1 Follower
 · 
86 Views
  • Source
    • "Western blots were quantified by calculating the background-normalized integrated intensity using ImageJ. Immunofluorescence was performed essentially as described in Keller et al. (2010). In brief, cells were plated on polylysine-coated cover glass, fixed in methanol at 20°C, blocked in 5% BSA, 1% fish gelatin, and 10% normal goat serum in PBS, and incubated with primary antibodies overnight (1:100). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.
    The Journal of Cell Biology 10/2012; 199(1):151-67. DOI:10.1083/jcb.201206068 · 9.69 Impact Factor
  • Source
    • "Cells bearing multiple motile cilia have a corresponding number of basal bodies, which equates to an excess of centrioles if these cells enter the cell cycle. Centrioles participate in mitotic spindle formation and the presence of too many centrioles can compromise the mitotic spindle (Keller et al., 2010). As such, cells bearing multiple cilia generally have a terminally differentiated and nonmitotic phenotype (Santos and Reiter, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Primary cilia are microscopic sensory antennae that cells in many vertebrate tissues use to gather information about their environment. In the kidney, primary cilia sense urine flow and are essential for the maintenance of epithelial architecture. Defects of this organelle cause the cystic kidney disease characterized by epithelial abnormalities. These findings link primary cilia to the regulation of epithelial differentiation and proliferation, processes that must be precisely controlled during epithelial repair in the kidney. Here, we consider likely roles for primary cilium-based signaling during responses to renal injury and ensuing epithelial repair processes.
    International review of cell and molecular biology 01/2012; 293:169-93. DOI:10.1016/B978-0-12-394304-0.00011-7 · 4.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The position of Chlamydomonas within the eukaryotic phylogeny makes it a unique model in at least two important ways: as a representative of the critically important, early-diverging lineage leading to plants, and as a microbe retaining important features of the last eukaryotic common ancestor (LECA) that have been lost in the highly studied yeast lineages. Its cell biology has been studied for many decades, and it has well-developed experimental genetic tools, both classical (Mendelian) and molecular. Unlike land plants, it is a haploid with very few gene duplicates, making it ideal for loss-of-function genetic studies. The Chlamydomonas cell cycle has a striking temporal and functional separation between cell growth and rapid cell divisions, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth with the cell division cycle; it also exhibits a highly choreographed interaction between the cell cycle and its centriole/basal body/flagellar cycle. Here we review the current status of studies of the Chlamydomonas cell cycle. We begin with an overview of cell cycle control in the well-studied yeast and animal systems, which has yielded a canonical, well-supported model. We discuss briefly what is known about similarities and differences in plant cell cycle control compared to this model. We next review the cytology and cell biology of the multiple fission cell cycle of Chlamydomonas. Lastly we review recent genetic approaches and insights into Chlamydomonas cell cycle regulation that have been enabled by a new generation of genomics-based tools. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Plant Journal 02/2015; 82(3). DOI:10.1111/tpj.12795 · 6.82 Impact Factor

Preview

Download
0 Downloads
Available from