Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position

Quantitative Analysis of Membrane Trafficking in Regulation of Cdc42 Polarity

Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: 1) determined that endocytosis plays an important role in Cdc42’s association with secretory vesicles 2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and 3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth

Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1

At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques

A cross-institutional analysis of the effects of broadening trainee professional development on research productivity

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Brandt, P. D., Sturzenegger Varvayanis, S., Baas, T., Bolgioni, A. F., Alder, J., Petrie, K. A., Dominguez, I., Brown, A. M., Stayart, C. A., Singh, H., Van Wart, A., Chow, C. S., Mathur, A., Schreiber, B. M., Fruman, D. A., Bowden, B., Wiesen, C. A., Golightly, Y. M., Holmquist, C. E., Arneman, D., Hall, J. D., Hyman, L. E., Gould, K. L., Chalkley, R., Brennwald, P. J., Layton, R. L. A cross-institutional analysis of the effects of broadening trainee professional development on research productivity. Plos Biology, 19(7), (2021): e3000956, https://doi.org/10.1371/journal.pbio.3000956.PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.Funding sources included the Common Fund NIH Director’s Biomedical Research Workforce Innovation Broadening Experiences in Scientific Training (BEST) Award. The following institutional NIH BEST awards (alphabetical by institution) included: DP7OD020322 (Boston University; AFB, ID, BMS, LEH); DP7OD020316 (University of Chicago; CAS); DP7OD018425 (Cornell University; SSV); DP7OD018428 (Virginia Polytechnic Institute; AVW, BB); DP7OD020314 (Rutgers University; JA); DP7OD020315 (University of Rochester; TB); DP7OD018423 (Vanderbilt University; KAP, AMB, KLG, RC); DP7OD020321 (University of California, Irvine; HS, DAF); DP7OD020317 (University of North Carolina, Chapel Hill; PDB, PJB, RLL); DP7 OD018427 (Wayne State University; CSC, AM). National Institutes of Health (NIH) General Medical Sciences - Science of Science Policy Approach to Analyzing and Innovating the Biomedical Research Enterprise (SCISIPBIO) Award (GM-19-011) - 1R01GM140282-01 (University of North Carolina at Chapel Hill; RLL, PDB, PJB)

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed