Roles of Hsl1p and Hsl7p in Swe1p degradation: beyond septin tethering.

The morphogenesis checkpoint in Saccharomyces cerevisiae couples bud formation to the cell division cycle by delaying nuclear division until cells have successfully constructed a bud. The cell cycle delay is due to the mitosis-inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. In unperturbed cells, Swe1p is degraded via a mechanism thought to involve its tethering to a cortical scaffold of septin proteins at the mother-bud neck. In cells that experience stresses that delay bud formation, Swe1p is stabilized, accumulates, and promotes a G(2) delay. The tethering of Swe1p to the neck requires two regulators, called Hsl1p and Hsl7p. Hsl1p interacts with septins, and Hsl7p interacts with Swe1p; tethering occurs when Hsl1p interacts with Hsl7p. Here we created a version of Swe1p that is artificially tethered to the neck by fusion to a septin so that Swe1p no longer requires Hsl1p or Hsl7p for its localization to the neck. We show that the interaction between Hsl1p and Hsl7p, required for normal Swe1p degradation, is no longer needed for septin-Swe1p degradation, supporting the idea that the Hsl1p-Hsl7p interaction serves mainly to tether Swe1p to the neck. However, both Hsl1p and Hsl7p are still required for Swe1p degradation, implying that these proteins play additional roles beyond localizing Swe1p to the neck.

Implementation of a responsible conduct of research education program at Duke University School of Medicine.

Academic medical centers rarely require all of their research faculty and staff to participate in educational programs on the responsible conduct of research (RCR). There is also little published evidence of RCR programs addressing high-profile, internal cases of misconduct as a way of promoting deliberation and learning. In the wake of major research misconduct, Duke University School of Medicine (DUSoM) expanded its RCR education activities to include all DUSoM faculty and staff engaged in research. The program included formal deliberation of the Translational Omics misconduct case, which occurred at Duke. Over 5,000 DUSoM faculty and staff participated in the first phase of this new program, with a 100% completion rate. The article reports on the program's development, challenges and successes, and future directions. This experience at Duke University illustrates that, although challenging and resource intensive, engagement with RCR activities can be integrated into programs for all research faculty and staff. Formal, participatory deliberation of recent cases of internal misconduct can add a novel dimension of reflection and openness to RCR educational activities.

Human cardiac-specific cDNA array for idiopathic dilated cardiomyopathy: sex-related differences.

Idiopathic dilated cardiomyopathy (IDCM) constitutes a large portion of patients with heart failure of unknown etiology. Up to 50% of all transplant recipients carry this clinical diagnosis. Female-specific gene expression in IDCM has not been explored. We report sex-related differences in the gene expression profile of ventricular myocardium from patients undergoing cardiac transplantation. We produced and sequenced subtractive cDNA libraries, using human left ventricular myocardium obtained from male transplant recipients with IDCM and nonfailing human heart donors. With the resulting sequence data, we generated a custom human heart failure microarray for IDCM containing 1,145 cardiac-specific oligonucleotide probes. This array was used to characterize RNA samples from female IDCM transplant recipients. We identified a female gene expression pattern that consists of 37 upregulated genes and 18 downregulated genes associated with IDCM. Upon functional analysis of the gene expression pattern, deregulated genes unique to female IDCM were those that are involved in energy metabolism and regulation of transcription and translation. For male patients we found deregulation of genes related to muscular contraction. These data suggest that 1) the gene expression pattern we have detected for IDCM may be specific for this disease and 2) there is a sex-specific profile to IDCM. Our observations further suggest for the first time ever novel targets for treatment of IDCM in women and men.

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint.

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.

Molecular dissection of the checkpoint kinase Hsl1p.

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.