Caenorhabditis elegans embryonic polarity requires the asymmetrically distributed proteins PAR-3, PAR-6 and PKC-3. The rho family GTPase CDC-42 regulates the activities of these proteins in mammals, flies and worms. To clarify its mode of action in C. elegans we disrupted the interaction between PAR-6 and CDC-42 in vivo, and also determined the distribution of GFP-tagged CDC-42 in the early embryo. Mutant PAR-6 proteins unable to interact with CDC-42 accumulated asymmetrically, at a reduced level, but this asymmetry was not maintained during the first division. We also determined that constitutively active GFP::CDC-42 becomes enriched in the anterior during the first cell cycle in a domain that overlaps with PAR-6. The asymmetry is dependent on PAR-2, PAR-5 and PAR-6. Furthermore, we found that overexpression of constitutively active GFP::CDC-42 increased the size of the anterior domain. We conclude that the CDC-42 interaction with PAR-6 is not required for the initial establishment of asymmetry but is required for maximal cortical accumulation of PAR-6 and to maintain its asymmetry.

PAR proteins play roles in the establishment and maintenance of polarity in many different cell types in metazoans. In C. elegans, polarity established in the one-cell embryo determines the anteroposterior axis of the developing animal and is essential to set the identities of the early blastomeres. PAR-1 and PAR-2 colocalize at the posterior cortex of the embryo. PAR-3, PAR-6 and PKC-3 (aPKC) colocalize at the anterior cortex of the embryo. A process of mutual exclusion maintains the anterior and posterior protein domains. We present results indicating that a homolog of the Hsp90 co-chaperone Cdc37 plays a role in dynamic interactions among the PAR proteins. We show that CDC-37 is required for the establishment phase of embryonic polarity; that CDC-37 reduction allows PAR-3-independent cortical accumulation of PAR-6 and PKC-3; and that CDC-37 is required for the mutual exclusion of the anterior and posterior group PAR proteins. Our results indicate that CDC-37 acts in part by maintaining PKC-3 levels and in part by influencing the activity or levels of other client proteins. Loss of the activities of these client proteins reveals that there are two sites for PAR-6 cortical association, one dependent on CDC-42 and not associated with PAR-3, and the other independent of CDC-42 and co-localizing with PAR-3. We propose that, in wild-type embryos, CDC-37-mediated inhibition of the CDC-42-dependent binding site and PAR-3-mediated release of this inhibition provide a key mechanism for the anterior accumulation of PAR-6.

BackgroundA proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.ResultsTo investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.ConclusionOverexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.

Polarity is a fundamental cellular feature that is critical for generating cell diversity and maintaining organ functions during development. In C. elegans, the one-cell embryo is polarized via asymmetric localization of the PAR proteins, which in turn are required to establish the future anterior-posterior axis of the embryo. PAR-3, a conserved PDZ domain-containing protein, acts with PAR-6 and PKC-3 (atypical protein kinase; aPKC) to regulate cell polarity and junction formation in a variety of cell types. To understand how PAR-3 localizes and functions during C. elegans development, we produced targeted mutations and deletions of conserved domains of PAR-3 and examined the localization and function of the GFP-tagged proteins in C. elegans embryos and larvae. We find CR1, the PAR-3 self-oligomerization domain, is required for PAR-3 cortical distribution and function only during early embryogenesis and that PDZ2 is required for PAR-3 to accumulate stably at the cell periphery in early embryos and at the apical surface in pharyngeal and intestinal epithelial cells. We also show that phosphorylation at S863 by PKC-3 is not essential in early embryogenesis, but is important in later development. Surprisingly neither PDZ1 not nor PDZ3 are essential for localization or function. Our results indicate that the different domains and phosphorylated forms of PAR-3 can have different roles during C. elegans development.

The TAS2R38 alleles that code for the PAV/AVI T2R38 proteins have long been viewed as benign taste receptor variants. However, recent studies have demonstrated an expanding and medically relevant role for TAS2R38. The AVI variant of T2R38 is associated with an increased risk of both colorectal cancer and Pseudomonas aeruginosa-associated sinus infection and T2R38 variants have been implicated in off-target drug responses. To address ethical concerns associated with continued student TAS2R38 gene testing, we developed an alternative to the traditional laboratory genotyping exercise. Instead of determining their own genotype, introductory level students isolated plasmid DNA containing a section of the human TAS2R38 gene from Escherichia coli. Following PCR-mediated amplification of a section of the TAS2R38 gene spanning the SNP at position 785, students determined their assigned genotype by restriction enzyme digestion and agarose gel electrophoresis. Using the course wide genotype and phenotype data, students found that there was an association between TAS2R38 genotype and the age of persistent P. aeruginosa acquisition in cystic fibrosis "patients." Assessment data demonstrated that students taking part in this new TAS2R38 laboratory activity made clear learning gains.

We describe a first-semester, integrated, introductory biology and chemistry course for undergraduates at Wellesley College in Wellesley, MA, USA. Our vision was to create a supportive learning community in which students could comfortably make connections between scientific disciplines as they learned necessary content for subsequent courses, further developed problem solving, communication, and laboratory skills, and meaningfully connected with other students and with faculty during their first semester in college. Through highlighting five guiding principles that are central to the course, we describe the integrated course structure and content as well as our efforts to build community, provide support, and engage students in building skills crucial to scientists. We also highlight features of this course and institutional policies that facilitated its logistical and collaborative implementation that can be adapted to fit the needs, goals, and constraints of a diverse range of institutions. A companion article describes an assessment of our course in achieving academic and community building goals.