The Scribble polarity protein stabilizes E-cadherin/p120-catenin binding and blocks retrieval of E-cadherin to the Golgi.
Department of Microbiology, Ctr for Cell Signaling, University of Virginia School of Medicine, Charlottesville, VA, USA.
Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins.
Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry.
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA.
Recruitment of dynein motors to the nuclear surface is an essential step for nucleus-centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2-Bicaudal D2 (BICD2) and Nup133- centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus-centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus-centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.
mASUN partially rescues spermatogenesis defects of Drosophila asun mutants. (A) Anti-CHY immunoblot of testes extracts from Drosophila wild-type (WT) males with or without germline expression of CHY-dASUN or CHY-mASUN; a relatively low expression level of a fusion protein of the expected size was observed for the latter. Tubulin was used as loading control. (B, C) Male fertility assays. “Rescue” indicates asun males with germline expression of CHY-mASUN, which increased the percentage of asun males producing progeny (B) and the average number of progeny per fertile male (C). (D–H) Germline CHY-mASUN expression restored perinuclear dynein in asun primary spermatocytes and spermatids. (D–F) Representative G2 spermatocytes stained for DHC and DNA are shown (D) with bar graphs depicting percentages of spermatocytes exhibiting perinuclear DHC (E) and average ratios of perinuclear to diffusely cytoplasmic DHC signal intensities (F). (G, H) Representative immature spermatids stained for DHC and DNA are shown (G) with bar graph depicting percentages of spermatids exhibiting perinuclear DHC (H). (I, J) Germline CHY-mASUN expression restored nucleus–centrosome coupling in asun primary spermatocytes. (I) Prophase I spermatocytes stained for β-tubulin and DNA. (J) Quantification of nucleus–centrosome coupling in prophase spermatocytes. **p < 0.0001, *p < 0.001. Scale bars, 50 μm.
Cell & Developmental Biology welcomes new faculty member Ken Lau, Ph.D. Dr. Ken Lau comes to Vanderbilt after completing his post postdoctorate training in the laboratory of Kenvi Hargis, Ph.D. In the Molecular Pathology Unit at the Massachusetts General Hospital and Harvard Medical School. Dr. Lau was chosen to join the Department of Cell and Developmental Biology because of his outstanding research qualifications and publication record, his unique background in experimental systems biology and computational biology, and his focus on intestinal epithelial function, which fit with the research missions of both the Epithelial Biology Center and the Department of Cell & Developmental Biology.
The central goal of my lab is to understand how inflammatory microenvironments affect epithelial cell behaviors in the context of human diseases, specifically, the role of the stem cell niche in colorectal cancer. Aberrant "stem cell-like" behavior is now recognized to contribute to the complexity of human diseases, for example, to therapeutic resistance and metastasis after conventional cancer therapies, which accounts for most of cancer-related fatalities. Devising ways to control these aberrant behaviors may lead to effective therapeutic strategies to combat these complex diseases. Why can't we simply eliminate these aberrant cells? Recent research shows that these aberrant cell populations are plastic. That is, differentiated cells can dynamically convert into stem-like cells and vice versa depending on environmental cues. Thus, instead of targeting a static cell type, the approach to manage these aberrant stem-like cells may be to control the environment where these cells are maintained.
In live tissues, cells must integrate dynamic mixtures of environmental cues through their signaling networks to arrive at response decisions. To understand the multivariate problem of how the microenvironment interacts with cells, we use multiplex and high throughput experimental approaches to characterize the network states (numbers and types of cells, secreted protein factors, intracellular signaling, and expressed genes, over time) within in vivo tissue. Our model system is the intestine of the laboratory mouse, whose state is controlled by interactions between the epithelium, immune system, and microbiota very much like in human. We then use the collected datasets over different experimental conditions to build data-driven mathematical models to quantitatively describe environment-cell input/output relationships and how these relationships are integrated to derive cellular outcomes. Because cell populations in in vivo tissues are not homogeneous, we will focus on generating data from single cells using techniques derived from flow cytometry and microscopy.
I received my training at Massachusetts General Hospital and MIT under joint supervision of Dr. Kevin Haigis (a mouse geneticist) and Dr. Douglas Lauffenburger (a bioengineer). There, I performed pioneering work on combining system-level signaling analyses and mouse models of intestinal diseases. I then further expanded these studies into looking at immune-epithelial cell interaction, and global signaling changes in mice with oncogenic mutations in the Ras proteins. Overall, I found that multivariate analyses are necessary for predicting signal-response relationships, whereas exploring single pathways one at a time is not predictive in vivo. I aim to foster a highly interdisciplinary and collaborative atmosphere in my lab, with a key focus on learning principles that will be translatable to human patients.
Ian G. Macara, Ph.D.
Louise B. McGavock and Chair, Department of Cell and Developmental Biology,
Vanderbilt University Medical School
Mary Heath, MBA, CFA, CRA, will assume the position of Departmental Administrator in the Department of Cell and Developmental Biology on April 22, 2013. Ms. Heath will be making the transition from Boston to Nashville as her new residence over the next few months. The department is anticipating the skills and experience that Mary will bring; anxious to add her leadership, grants experience and financial acumen to our administrative team here at Vanderbilt and in our department.
Please join us in welcoming Mary to Vanderbilt University Medical School and the Department of Cell and Developmental Biology.
Mary K. Heath, MBA, CFA, CRA
Ms. Heath has over ten years of academic administrative leadership experience in managing and overseeing administrative/operational
responsibilities in a scientific research setting. She has a degree in Bio-medical Engineering from Brown University and earned her MBA from Simmons College while working full time. She is also a certified financial analyst and research administrator.
Ms. Heath has for the last six years been the Department Administrator of the Department of Cancer Immunology and AIDS at the Dana Farber Cancer Institute, Harvard Medical School. She directed the administrative and operational responsibilities for a department consisting of 200 employees, with fourteen faculty and research funding in excess of $24 million dollars annually, including 2 NIH training grants.
Ms. Heath had previously worked for the Heller School for Social Policy and Management at Brandeis University, as their Associate Director of Grants and Contracts Management. She has a very strong background in finance and business practices, having spent her early career in investment management, as assistant Vice-President of MFS Investment Management, Inc. and Chief Financial Officer for Applied Value Corporation.
M.B.A., Simmons College, 1988
B.A., Biomedical Engineering, Brown University, 1982
Lucy X. Lua, Maria Rosa Domingo-Sananesb, Malwina Huzarskaa, Bela Novakb, and Kathleen L. Gould a,c,1
+ Author Affiliations
a Department of Cell and Developmental Biology, and
c The Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37212; and
b Oxford Centre for Integrative Systems Biology, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
Edited by Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved May 4, 2012 (received for review January 26, 2012)
Cyclin-dependent kinase 1 (Cdk1) kinase dephosphorylation and activation by Cdc25 phosphatase are essential for mitotic entry. Activated Cdk1 phosphorylates Cdc25 and other substrates, further activating Cdc25 to form a positive feedback loop that drives the abrupt G2/mitosis switch. Conversely, mitotic exit requires Cdk1 inactivation and reversal of Cdk1 substrate phosphorylation. This dephosphorylation is mediated, in part, by Clp1/Cdc14, a Cdk1-antagonizing phosphatase, which reverses Cdk1 phosphorylation of itself, Cdc25, and other Cdk1 substrates. Thus, Cdc25 phosphoregulation is essential for proper G2–M transition, and its contributions to cell cycle control have been modeled based on studies using Xenopus and human cell extracts. Because cell extract systems only approximate in vivo conditions where proteins interact within dynamic cellular environments, here, we use Schizosaccharomyces pombe to characterize, both experimentally and mathematically, the in vivo contributions of Cdk1-mediated phosphorylation of Cdc25 to the mitotic transition. Through comprehensive mapping of Cdk1 phosphosites on Cdc25 and characterization of phosphomutants, we show that Cdc25 hyperphosphorylation by Cdk1 governs Cdc25 catalytic activation, the precision of mitotic entry, and unvarying cell length but not Cdc25 localization or abundance. We propose a mathematical model that explains Cdc25 regulation by Cdk1 through a distributive and disordered phosphorylation mechanism that ultrasensitively activates Cdc25. We also show that Clp1/Cdc14 dephosphorylation of Cdk1 sites on Cdc25 controls the proper timing of cell division, a mechanism that is likely due to the double negative feedback loop between Clp1/Cdc14 and Cdc25 that controls the abruptness of the mitotic exit switch.
Cyclin-dependent kinases (CDKs) are key regulators of the eukaryotic cell cycle. At mitotic entry, the Cdc25 family phosphatases activate Cdk1-CyclinB complexes by removing inhibitory phosphorylations on Cdk1 catalyzed by Wee1 family kinases. Activated Cdk1-CyclinB phosphorylates its substrates and drives mitotic entry (1, 2). Cell cycle modeling showed that a bistable trigger facilitates the switch-like transition between interphase and mitosis (3⇓–5). Bistability ensures that there can only be two stable steady states for the system (interphase or mitosis); it predicts a Cdk1 activity threshold for mitotic entry and a lower activity threshold for mitotic exit, thus giving rise to hysteresis in the system.
Xenopus laevis and human cell extract studies found that the bistable mitotic switch is modulated by at least two feedback loops: the Cdk1-Wee1 double negative feedback loop, in which Cdk1 and Wee1 inactivate one another by phosphorylation, and the Cdk1-Cdc25 positive feedback loop, where Cdk1 phosphorylates and activates Cdc25, while Cdc25 dephosphorylates and further activates Cdk1 (3⇓⇓⇓–7). In addition to a positive or double negative feedback loop, bistability requires an ultrasensitive response of at least one component of a feedback loop (8, 9). It has been proposed that Cdc25 activation and Wee1 inactivation by Cdk1 are ultrasensitive in nature; their activity follows sigmoid signal response curves (rising and decreasing, respectively) as a function of Cdk1 activity (10, 11). In Xenopus egg extracts, ultrasensitivity in Wee1 inactivation is attributed to competition between essential and nonessential Cdk1 phosphosites on Wee1 and between Wee1 and other Cdk1 substrates (6). In addition, Cdk1 multisite phosphorylation of XCdc25C contributes to its ultrasensitive activation (7). Although ex vivo and mathematical models suggest that both Cdk1-Wee1 and Cdk1-Cdc25 feedback loops contribute to the robustness of the mitotic entry switch, perturbation of the feedback loops in vivo in cycling cells has yet to be analyzed.
Mitotic exit and the spindle assembly checkpoint may also be modulated by a bistable switch (12⇓–14). In Saccharomyces cerevisiae, Cdc14, a phosphatase that dephosphorylates Cdk1 substrates (15, 16), adds abruptness to the metaphase–anaphase switch by interacting with Securin, a protein that protects sister chromatid separation until anaphase onset in an ultrasensitive positive feedback loop. Cdc14 dephosphorylates Securin to target it for ubiquitylation and degradation. Degradation of Securin activates Separase, which also activates Cdc14 (13). Our laboratory and other groups found that Clp1, the Schizosaccharomyces pombe Cdc14 ortholog, dephosphorylates Cdc25 on Cdk1 phosphosites, and this dephosphorylation correlates with Cdc25 inactivation and degradation (17, 18). Because Cdc25 activates Cdk1, the activity of which inhibits Clp1 activity (19), the interaction between Clp1 and Cdc25 may form a feedback loop that contributes to the mitotic exit switch in S. pombe.
Here, we use S. pombe to further understand how Cdc25 phosphorylation by Cdk1 contributes to the mitotic entry and exit switches in cycling cells. Using this in vivo model, we suggest a mechanism of direct Cdk1 activation and Clp1 inactivation of Cdc25. Also, we find that the Cdk1-Cdc25 positive feedback loop is important for the precision of mitotic entry and maintenance of uniform cell length. Finally, we suggest that the interactions of Clp1, Cdk1, and Cdc25 create a double negative feedback loop that significantly contributes to the robustness of mitotic exit, specifically controlling the timing of cell division.
Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis
Poojitha Sitaram, Michael A. Anderson*, Jeanne N. Jodoin, Ethan Lee and Laura A. Lee‡
+ Author Affiliations
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, U-4225 Medical Research Building III, 465 21st Avenue South, Nashville, TN 37232-8240, USA.
+ Author Notes
* Present address: Georgetown University Law Center, 600 New Jersey Avenue NW, Washington, DC 20001, USA
‡ Author for correspondence (firstname.lastname@example.org)
Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, we characterized a male-sterile allele of the Drosophila homolog of human LIS1. We found that centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, we observed loss of attachments between the nucleus, basal body and mitochondria. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. We show that dynein recruitment to the nuclear surface and spindle poles is severely reduced in Lis-1 male germ cells. We propose that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, as we observed similar phenotypes in flies null for Tctex-1, a dynein light chain. We have previously identified asunder (asun) as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis. We now report that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. We found that Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. We present a model in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis.
The fission yeast septation initiation network (SIN) kinase, Sid2, is required for SIN asymmetry and regulates the SIN scaffold, Cdc11
Anna Feoktistovaa, Jennifer Morrell-Falveya, Jun-Song Chena, N. Sadananda Singh, Mohan K. Balasubramanianb,c, and
Kathleen L. Goulda,1
aHoward Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37212
bTemasek Life Sciences Laboratory, National University of Singapore, Republic of Singapore 117604
cMechanobiology Institute and Department of Biological Sciences, National University of Singapore, Republic of Singapore 117604
Rong Li, Monitoring Editor
Submitted September 19, 2011.
Revised February 7, 2012.
Accepted March 9, 2012.
The Schizosaccharomyces pombe septation initiation network (SIN) is an Spg1-GTPase–mediated protein kinase cascade that triggers actomyosin ring constriction, septation, and cell division. The SIN is assembled at the spindle pole body (SPB) on the scaffold proteins Cdc11 and Sid4, with Cdc11 binding directly to SIN signaling components. Proficient SIN activity requires the asymmetric distribution of its signaling components to one of the two SPBs during anaphase, and Cdc11 hyperphosphorylation correlates with proficient SIN activity. In this paper, we show that the last protein kinase in the signaling cascade, Sid2, feeds back to phosphorylate Cdc11 during mitosis. The characterization of Cdc11 phosphomutants provides evidence that Sid2-mediated Cdc11 phosphorylation promotes the association of the SIN kinase, Cdc7, with the SPB and maximum SIN signaling during anaphase. We also show that Sid2 is crucial for the establishment of SIN asymmetry, indicating a positive-feedback loop is an important element of the SIN.