We have recently shown that the ADP-ribosyltransferase activity of P. aeruginosa
ExoT toxin inhibits late stages of cytokinesis.10
As Crk adapter proteins are the primary targets of ExoT,11
we tested the hypothesis that Crk proteins are involved in cytokinesis. The studies reported in this communication confirm our hypothesis and reveal the requirement for other focal adhesion proteins in mammalian cytokinesis.
Despite lack of previous data implicating Crk in cytokinesis, we provide compelling evidence that Crk function is essential to this process. We propose that Crk is required at steps after the completion of the cleavage furrow and is a prerequisite for the terminal steps of cytokinesis. This interpretation is supported by the findings that (i) Crk DN mutants inhibit cytokinesis post-cleavage furrow formation; (ii) Crk is detectable at the midbody after cleavage furrow; and that (iii) siRNA depletion of Crk prevents syntaxin-2 midbody localization, a requirement for the final step of cytokinesis, without affecting RhoA or citron K localization to the midbody, events which occur prior to ingression and are required for earlier steps.
Tyrosine phosphorylation of paxillin by Src tyrosine kinase and the subsequent phospho-paxillin recruitment of Crk are among the common features that underlie the Crk-mediated signaling cascades in cellular processes, such as focal adhesion assembly and cell migration.20
Our data suggest that similar strategy may be used in cytokinesis. We now show that phospho-paxillin is also present at the midbody and that paxillin also serves an essential role in cytokinesis. Our data demonstrate that paxillin mediates intermediate steps in cytokinesis, post RhoA-mediated cleavage furrowing and prior to Crk midbody localization which occurs after furrow completion. Paxillin siRNA depletion prevents Crk enrichment at the midbody but has no effect on midbody localization of RhoA and citron K. As would be expected from a protein that is required for Crk localization to the midbody, paxillin is also essential for syntaxin-2 midbody recruitment during cytokinesis. The requirement of paxillin for Crk midbody localization is consistent with its role in recruiting Crk in other cellular contexts such as focal adhesion assembly and cell migration.20
The involvement of paxillin in cytokinesis is further supported by recent reports demonstrating that Pxl1p, a paxillin homolog in the fission yeast Schizosaccharomyces pombe
, plays a major role in cytokinesis.29
In our studies reported here, depletion of Paxillin or Crk by siRNA or the expression of Crk DN mutants in HeLa cells did not grossly affect the cell matrix interaction yet inhibited cytokinesis. Taken together, these data suggest that the role of paxillin and Crk in cytokinesis is likely independent of their role in focal adhesion assembly and cell migration or that focal adhesion as a unit may partake in this cellular process.
Src is a known tyrosine kinase for paxillin.15
While it has been demonstrated that Src activity is required to initiate furrowing25
and to dissociate daughter cells during the final step in cytokinesis, abscission,24
our data further expand the role of Src family kinases in cytokinesis. We demonstrate that Src localizes to the midbody early during cytokinesis and that its kinase activity is also required for cleavage furrow completion during cytokinesis. Although our data indicate that Src is a major contributor to paxillin phosphorylation in HeLa cells, more studies are needed to definitively address whether paxillin phosphorylation at the midbody is Src-dependent and critical for recruitment of Crk to this compartment.
Genetic screens in Drosophila and C. elegans
, together with proteomic analysis of purified mammalian midbodies, have led to the identification of many proteins that contribute to cytokinesis, including vinculin, talin and enigma, components of focal adhesions, but failed to identify Crk or paxillin.2
It should be noted that these methods also failed to identify other essential components required late in cytokinesis, including syntaxin-2, endobrevin, Cep55, centriolin and the exocyst components, highlighting the need for multipronged approaches to dissect the molecular mechanisms of cytokinesis.
Since syntaxin-2 lacks identifiable Crk interacting domains, it is unlikely that Crk recruits syntaxin-2 directly to midbody. Indeed, we have been unable to co-immunoprecipitate syntaxin-2 or its partner endobrevin with Crk or paxillin (unpublished results). Other Crk and/or paxillin interacting proteins may recruit syntaxin-2 to the midbody. Intriguingly, Crk interacts with pleckstrin homology domain (PH)- containing proteins such as DHR and Gab.22
These proteins interact with membrane inositol phospholipids.31
Thus, the DHR and/or Gab proteins, in association with Crk, could potentially mediate the recruitment of membrane-associated exocyst and syntaxin-2 proteins to the midbody during cytokinesis. More experiments are needed to test this hypothesis.
Based on their critical roles in cytokinesis, Crk or paxillin deficient mice would be expected to manifest an embryonic lethal phenotype. Indeed, attempts to construct mice deficient in both CrkI and CrkII have failed, while CrkII null mice are viable,33
indicating that CrkI may be sufficient for all Crk-associated functions including cytokinesis. In contrast, paxillin deficient embryos develop normally until dying at gastrulation (E6.5-E7.5),34
interestingly a time at which paxillin begins to be expressed during normal mouse development.34
Prior to this time point, Hic-5 and leupaxin, two closely related paxillin family members,35
and potentially could compensate for the absence of paxillin in cytokinesis and other critical processes. Interestingly, paxillin versus Hic-5 and leupaxin exhibit mutually exclusive tissuespecific expression. While paxillin is expressed in all epithelial cell lines examined thus far, Hic-5 and leupaxin expression have not been detected in epithelial tissues or cell lines, including HeLa cells,35
providing an explanation for the requirement for paxillin in cytokinesis in HeLa cells.
The hierarchical order of assembly of midbody components and their interactions remain largely unknown. The presence of hundreds of proteins in this compartment, however, highlights the need for adaptor proteins to act as molecular glue during cytokinesis. Scaffolding proteins such as paxillin and Crk possess multiple protein interaction domains12
and are highly suited to function as centers for the assembly of the multi-protein signaling and structural complexes at the midbody.
A growing body of evidence suggest that traction forces and/or cell migration may contribute to cytokinesis. Traction forces have been suggested to contribute to cytokinesis in dividing amoebae.40
During furrowing and midbody extension in dividing human fibroblasts, force is generated,41
supporting the notion that traction-mediated cell migration may contribute to daughter-cell scission.41
Since Crk, paxillin and Src play pivotal role in cell migration, it is intriguing to postulate that in addition to their role in recruiting syntaxin-2 and perhaps other essential components of late cytokinetic apparatus, Crk and paxillin may also indirectly contribute to cytokinesis by providing traction-mediated force during abscission. We are currently conducting research aimed at examining this hypothesis.
In summary, our results demonstrate that paxillin and Crk play key roles in late stages of cytokinesis. Our findings provide a solid foundation to further elucidate the role of focal adhesion components in this fundamental biological process.