Robert L. Margolis

G1 tetraploidy checkpoint and the suppression of tumorigenesis

Checkpoints suppress improper cell cycle progression to ensure that cells maintain the integrity of their genome. During mitosis, a metaphase checkpoint requires the integration of all chromosomes into a metaphase array in the mitotic spindle prior to mitotic exit. Still, mitotic errors occur in mammalian cells with a relatively high frequency. Metaphase represents the last point of control in mitosis. Once the cell commits to anaphase there are no checkpoints to sense segregation defects. In this context, we will explore our recent finding that non‐transformed mammalian cells have a checkpoint that acts subsequent to mitotic errors to block the proliferation of cells that have entered G1 with tetraploid status. This arrest is dependent upon both p53 and pRb, and may represent an important function of both p53 and pRb as tumor suppressors. Further, we discuss the possibility that this mechanism may similarly impose G1 arrest in cells that become aneuploid through errors in mitosis.

Crystal Structure of Human Survivin Reveals a Bow Tie–Shaped Dimer with Two Unusual α-Helical Extensions

Abstract Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie–shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein–protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its α-helical extensions.

MICROTUBULE TREADMILLING : WHAT GOES AROUND COMES AROUND

“Ill see it when I believe it”u2003Daniel Mazia

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression.

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

Colocalization of TD-60 and INCENP throughout G2 and mitosis: evidence for their possible interaction in signalling cytokinesis.

Abstract.u2002TD-60 and INCENP are two members of the chromosome passenger protein family, and each has been suggested to play a role in the control of cytokinesis. Here we demonstrate by confocal immunofluorescence microscopy that TD-60 and INCENP distribute identically throughout the cell cycle. Both appear coordinately in G2-phase nuclei and become concentrated at centromeres during prophase. TD-60 and INCENP both then leave the chromosome together during anaphase and redistribute to the spindle midzone, as do other chromosome passenger proteins, and traverse the entire equatorial diameter from cortex to cortex. By image overlay and pixel count analysis we show that TD-60 and INCENP are distinct among known chromosome passenger proteins in extending beyond the spindle to the cortex. Further, we show that the cytokinesis-associated protein kinase AIM-1 also shares this distribution property. We suggest that this redistribution is required to signal cytokinesis. TD-60 and INCENP also show identical localization in cells that exit mitosis in the presence of dihydrocytochalasin B (DCB), an inhibitor of actin assembly. Such cells can resume cleavage upon removal of DCB and in a recovery subpopulation that cleaves only on one side, these proteins both colocalize to the cortex only where a cleavage furrow forms. Given the coincident distribution of TD-60 and INCENP during both interphase and mitosis, we suggest that these proteins may cooperate, perhaps within a protein complex, in signalling cytokinesis. Such a mechanism, using chromosome passenger proteins, may ensure that cytokinesis occurs only between the separated chromatids, and only after they have segregated.

Molecular Distinctions between Aurora A and B: A Single Residue Change Transforms Aurora A into Correctly Localized and Functional Aurora B

Aurora A and Aurora B, paralogue mitotic kinases, share highly similar primary sequence. Both are important to mitotic progression, but their localizations and functions are distinct. We have combined shRNA suppression with overexpression of Aurora mutants to address the cause of the distinction between Aurora A and Aurora B. Aurora A residue glycine 198 (G198), mutated to asparagine to mimic the aligned asparagine 142 (N142) of Aurora B, causes Aurora A to bind the Aurora B binding partner INCENP but not the Aurora A binding partner TPX2. The mutant Aurora A rescues Aurora B mitotic function. We conclude that binding to INCENP is alone critical to the distinct function of Aurora B. Although G198 of Aurora A is required for TPX2 binding, N142G Aurora B retains INCENP binding and Aurora B function. Thus, although a single residue change transforms Aurora A, the reciprocal mutation of Aurora B does not create Aurora A function. An Aurora A-Delta120 N-terminal truncation construct reinforces Aurora A similarity to Aurora B, because it does not associate with centrosomes but instead associates with kinetochores.

Distinct Dynamics of Aurora B and Survivin during Mitosis

We have studied the dynamics of Aurora B and Survivin during mitosis in living cells, using C-terminal GFP chimeras of the two proteins. These chimeras showed identical localization and behave as bona fide wild type proteins. The mobility of Aurora B-GFP and Survivin-GFP was analyzed by FRAP. The data show that Survivin-GFP, in contrast to Aurora B-GFP, is highly mobile at prometaphase and metaphase. At telophase and cell cleavage, both chimeras are found to be fully immobile. The ablation of Aurora B by siRNA results in a dramatic decrease of the Survivin-GFP mobility. These results demonstrate that Survivin, but not Aurora B, is weakly associated with the centromeric chromatin at prometaphase and metaphase. The weak association of Survivin with centromeric chromatin is dependent on the presence of Aurora B and is not affected by treatment with either nocodazole or taxol. The rapid and conditional interchange between passenger proteins that we show by live imaging indicates that the high affinity interactions demonstrated with in vitro analysis of passenger protein binding are, in fact, static “snapshots” of highly dynamic and regulated in vivo interactions in mitotic cells.

The Role of the PH Domain and SH3 Binding Domains in Dynamin Function

Dynamin, a 100 kD GTPase, is necessary for the normal development and function of mammalian neural tissue. In neurons, it is necessary for the biogenesis of synaptic vesicles, and in other cell types dynamin has a general and important role in clathrin mediated receptor endocytosis. Different isoforms function as molecular scissors either during the formation of coated vesicles from plasma membrane coated pits, or during the release of intracellular vesicles from donor membranes. The mechanism entails the formation of a horseshoe-shaped dynamin polymer at the neck of the budding vesicle, followed by neck scission through a GTP hydrolysis dependent activity. The primary sequence of dynamin contains several C-terminal SH3 binding proline motifs, a central pleckstrin homology (PH) domain, and an N-terminal GTPase domain. Each of these domains appears to play a distinct role in dynamin function. Dynamin is activated by stimulus coupled PKC phosphorylation in brain, possibly mediated through PKC interactions with the PH domain. Further, SH3 domain interactions with the C-terminal sequences and phophatidylinositol/G beta gamma interactions with the PH domain also increase dynamin GTPase activity. Each of these various regulatory mechanisms is important in dynamin function during vesicle budding, although the means by which these mechanisms integrate in the overall function of dynamin remains to be elucidated.

Analysis of the Spindle-Assembly Checkpoint in HeLa Cells

The spindle-assembly checkpoint involves signaling at kinetochores, which leads to the arrest of mitotic progression in the absence of microtubule attachment or spindle tension. Here, we detail procedures for the analysis of the spindle-assembly checkpoint in adherent mammalian cells. These techniques focus on pharmacological approaches and immunofluorescence microscopy to verify the state of spindle assembly, kinetochore attachment of microtubules and spindle tension, chromosome positioning, and kinetochore signaling by the Mad2 or Bub1 checkpoint proteins. We also describe a bi-parameter flow cytometric assay, using either MPM-2 or anti-phospho-(Ser10)-histone H3 antibodies, for quantitating mitotic cells.