Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269-4156, USA
Address correspondence to Joe LoTurco, Associate Professor, Physiology and Neurobiology, 3107 Horsebarn Hill Road, U4156, University of Connecticut, Storrs, CT 06269-4156, USA. Email: Loturco{at}uconn.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Citron kinase was initially identified in a screen for genes with similarity to the kinase domain of Rho-activated kinases (ROCK and ROK kinases), downstream effectors of Rho family GTPases (Di Cunto et al., 1998; Madaule et al., 1998
). In a functional test, C-terminal truncation mutants of citron kinase were shown to cause erratic cytokinetic behaviors and cytokinesis failure when overexpressed in Hela-cells (Madaule et al., 2000
). These gain-of-function experiments suggested that citron kinase is essential to stabilizing the position and ensuring normal function of the cytokinesis furrow and midbody. More recent analyses of a citron kinase null mutant mouse (Di Cunto et al., 2000
) and of the flathead mutation in rat (Cogswell et al., 1998
; Sarkisian et al., 2002
) further confirmed that citron kinase is essential to cytokinesis in vivo, and further showed that this requirement is specific for CNS precursors.
The expression pattern of citron kinase through embryonic and perinatal development suggests that it is essential for many embryonic and postnatally generated neurons but not for postnatally generated glia. Citron kinase message and protein is expressed across the VZ; however, by postnatal day 1 citron kinase expression is absent in the forebrain but still present in the cerebellum throughout the period of granule cell neuronogenesis (Di Cunto et al., 2000; Sarkisian et al., 2002
). Similarly, immunocytochemistry and Western blot analyses show that citron kinase is not expressed in the sub-ventricular zone in postnatal cortex (unpublished observation).
Considering the relative specificity of citron kinase to neurogenic cell divisions, it becomes important to define the signaling systems that engage citron kinase during neurogenesis. Based both on amino acid sequence and functional studies, citron kinase protein has multiple functional domains that can interact with other signaling and structural proteins (Di Cunto et al., 1998; Madaule et al., 1998
; Zhang et al., 1999
; Eda et al., 2001
). The amino terminal kinase domain shows significant homology to ROCK kinases and phosphorylates regulatory myosin light chain (RMLC) (Matsumura et al., 2001
). The C-terminal contains many protein interaction domains including a coiled-coil domain, Rho binding domain, PDZ-binding domain, zinc finger domain, and putative SH3-binding domain (Madaule et al., 1998
). In addition to multiple protein interaction domains, citron kinase moves to different cellular compartments through mitosis (Eda et al., 2001
). Citron kinase moves from the cytoplasm to the spindle midzone, and then is transported in telophase to the cytokinesis furrow in a Rho GTPase dependent fashion. This dynamic localization through the cell cycle and multi-domain structure of citron kinase suggest that it may have multiple functions during neurogenic cell divisions.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous reports have shown that citron kinase null mutants have binucleate neurons throughout the CNS. We have used multiple neuronal and glial cell markers to determine the proportions of different types of neurons and glia that are binucleate. As shown in Figure 1, neuronal markers label binucleate neurons that are positive for GABA, and several other markers including NeuN, Pyramidal cell marker (Swant), and doubleortin (not shown). In a previous study we also showed that many cells that label with interneuron markers are also binucleate. In contrast to the neuronal markers, glial markers, GFAP and NG2, a marker for oligodendrocyte precursors, do not label significant numbers of binucleate cells. In the flathead mutant rat from P0P21, while nearly 50% of cells that are positive for neuronal markers are binucleate, only 3% of GFAP+ (n = 500 cells, four animals) and 0% of NG2+ cells are binucleate (out of 700 cells, in four animals). Therefore, citron kinase appears to be essential for normal completion of cell divisions that produce neurons, but not for most of the cell divisions that produce glial cells. This phenotype is also consistent with the temporal expression pattern of citron kinase, which is expressed in prenatal periods in the neocortex but not postnatally when the majority of gliogenic cell divisions in cortex occur.
Citron Kinase Is Essential for Mitosis
In cell lines, citron kinase has been shown to initially localize to the spindle midzone during anaphase and then move to the cytokinesis furrow and midbody after activation by Rho GTPase (Eda et al., 2001). To date, the analysis of citron kinase mutants has indicated an essential role during cytokinesis, and a potential role for citron kinase during mitosis when citron kinase is present at the spindle midzone has not been previously examined. To test the hypothesis that citron kinase plays a role during mitosis we compared time-lapse images from E17 and E15 VZ explants from wild-type and homozygous mutant flathead embryos (fh/fh). Images were taken form four wild-type and four mutant embryos at E17 and two wild-type and mutant embryos at E15 in a plane tangential to the surface of the VZ. This explant preparation enabled us to image multiple mitoses in a single plane without cell behavior being affected by cut edges of a slice. The majority of metaphase plates rotate in a plane parallel to the plane of the image, and
5% were seen to rotate in a plane perpendicular to the plane of the VZ surface in this preparation. Metaphase plates stopped rotating just prior to entering anaphase, and chromosome separation was completed most typically within 15 min (two frames) after cessation of metaphase plate rotation. In wild-type explants, metaphase plates divided symmetrically through the length, and were never observed to separate incompletely or asymmetrically. In addition, while some anaphases appeared to be delayed after the formation of a metaphase plate, no metaphase plates in wild-type were observed not to enter anaphase within a 2 h imaging session.
In contrast to images of mitoses in wild-type, time lapse imaging of mitotic behavior of cells at the VZ surface of flathead mutants indicated many disrupted mitoses (Fig. 2, movie in supplementary data). Metaphase plates formed normally in most mitotic cells in fh/fh; however,
50% of cells in flathead either did not progress into anaphase or showed disrupted patterns of chromosome segregation during anaphase. As shown in Figure 2
, these abnormalities could be grouped generally into two different categories. Approximately 20% of mitoses in mutants were either blocked or greatly slowed in progression from metaphase to anaphase (Fig. 2b
). These cells formed mitotic plates but over at least 2 h of imaging never progressed into anaphase as assessed by chromosome separation. At the edge of the metaphase plates in these cells there were often stray chromosomes that were seen to move in and out of the metaphase plate. Approximately 20% of mitoses in mutant at E15 and 30% at E17 showed disruptions in anaphase that were characterized as aberrant because of altered or failed chromosome separation (Fig. 2
ce). Since chromosome separation primarily requires the normal function of the spindle apparatus, we interpret the patterns of disrupted mitoses in the flathead mutant as disruptions in spindle function. Normal spindle function during cell division at the VZ surface is therefore dependent upon citron kinase.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is not currently known why loss of citron kinase affects only a subset of progenitors. ROCK kinases are present within VZ cells and are the most likely candidates for compensating for citron kinase function (Di Cunto et al., 2000). Alternatively, cell division in some progenitors may be independent of Rho and associated kinases. For example, inhibition of Rho has been shown to disrupt cytokinesis of weekly adherent cells in culture but not strongly adherent cells, suggesting that Rho may not be essential for division of cells in certain functional states (OConnell et al., 1999
).
Why do different progenitors require different cell division mechanisms? One possibility is that different fates are generated preferentially from different types of cell divisions. Such a suggestion has been previously made for cortex (Adams, 1996; OConnell et al., 1999
; Miyata et al., 2001
; Noctor et al., 2001
) and in Drosophila, orientation of division has been shown to determine neurogenic fates (Knoblich et al., 1995
; Lin and Schagat, 1997
). We have not observed a quantitative difference in the orientation of cell divisions in flathead, however (unpublished observation), so it seems unlikely that different cytokinesis mechanisms revealed by analysis of the flathead mutant are related to different division planes. Nevertheless, some cell types are disproportionately affected in flathead mutants. Upper layer pyramidal neurons, for example, are nearly missing in flathead and similarly cortical inhibitory interneurons are severely depleted in all layers (Roberts et al., 2000
; Sarkisian et al., 2001
). This suggests that there may be different mechanisms for mitoses that generate different cell types in cortex. By determining the function of different proteins in the signaling pathway associated with citron kinase it may be possible to determine a more detailed relationship between mechanisms of cell division and the generation of different neocortical cell types.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cogswell CA, Sarkisian MR, Leung V, Patel R, DMello SR, LoTurco JJ (1998) A gene essential to brain growth and development maps to the distal arm of rat chromosome 12. Neurosci Lett 251:58.
Di Cunto F, Calautti E, Hsiao J, Ong L, Topley G, Turco E, Dotto GP (1998) Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the RhoRac-binding protein Citron. J Biol Chem 273:2970629711.
Di Cunto F, Imarisio S, Hirsch E, Broccoli V, Bulfone A, Migheli A, Atzori C, Turco E, Triolo R, Dotto GP, Silengo L, Altruda F (2000) Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron 28:115127.[ISI][Medline]
Eda M, Yonemura S, Kato T, Watanabe N, Ishizaki T, Madaule P, Narumiya S (2001) Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells. J Cell Sci 114:32733284.[ISI][Medline]
Knoblich JA, Jan LY, Jan YN (1995) Asymmetric segregation of Numb and Prospero during cell division. Nature 377:624627.[CrossRef][ISI][Medline]
Komatsu S, Yano T, Shibata M, Tuft RA, Ikebe M (2000) Effects of the regulatory light chain phosphorylation of myosin II on mitosis and cytokinesis of mammalian cells. J Biol Chem 275:3451234520.
Lin H, Schagat T (1997) Neuroblasts: a model for the asymmetric division of stem cells. Trends Genet 13:3339.[CrossRef][ISI][Medline]
Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H, Ishizaki T, Narumiya S (1998) Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 394:491494.[CrossRef][ISI][Medline]
Madaule P, Furuyashiki T, Eda M, Bito H, Ishizaki T, Narumiya S (2000) Citron, a Rho target that affects contractility during cytokinesis. Microsc Res Tech 49:123126.[CrossRef][ISI][Medline]
Matsumura F, Totsukawa G, Yamakita Y, Yamashiro S (2001) Role of myosin light chain phosphorylation in the regulation of cytokinesis. Cell Struct Funct 26:639644.[CrossRef][ISI][Medline]
Miyata T, Kawaguchi A, Okano H, Ogawa M (2001) Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31:727741.[ISI][Medline]
Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409:714720.[CrossRef][ISI][Medline]
OConnell CB, Wheatley SP, Ahmed S, Wang YL (1999) The small GTP-binding protein rho regulates cortical activities in cultured cells during division. J Cell Biol 144:305313.
Roberts MR, Bittman K, Li WW, French R, Mitchell B, LoTurco JJ, DMello SR (2000) The flathead mutation causes CNS-specific developmental abnormalities and apoptosis. J Neurosci 20:22952306.
Sarkisian MR, Frenkel M, Li W, Oborski JA, LoTurco JJ (2001) Altered interneuron development in the cerebral cortex of the flathead mutant. Cereb Cortex 11:734743.
Sarkisian MR, Li W, Di Cunto F, DMello SR, LoTurco JJ (2002) Citron-kinase, a protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant rat. J Neurosci 22:RC217.[CrossRef][Medline]
Zhang W, Vazquez L, Apperson M, Kennedy MB (1999) Citron binds to PSD-95 at glutamatergic synapses on inhibitory neurons in the hippocampus. J Neurosci 19:96108.