Customized Virtual Simulations Provide an Interactive Lab Experience.

Although various resources exist for facilitating online laboratory courses, stitching together disparate elements from multiple sources may not be sufficient to meet the learning goals of a given course. For example, our Biology Project Lab course introduces students to an array of fundamental laboratory techniques, and the COVID-19 pandemic necessitated the development of virtual laboratory options for remote learners. We anticipated that the logic and application of the course material-a multiday sequence of connected experiments-would be lost if we combined prefabricated labs from a variety of sources. Moreover, we wanted students to familiarize themselves with our laboratory equipment, while providing interactive experiences rather than passive video demonstrations. Therefore, we used Storyline 360 to create a series of interactive lab modules to accommodate students who were remote or in quarantine. These online labs were integrated with our learning management system (LMS) and included exercises such as video demonstrations, short answer responses, image selection, drag-and-drop activities, and organizing procedural steps. Our simulations can be shared with instructors and customized for their own interactive labs, or instructors can build course-specific modules from scratch using the Storyline 360 platform. Although the simulations could not fully replicate the in-person learning experience, students appreciated being able to watch and participate in lab activities and recommended that the labs be retained as supplemental activities in future semesters. Storyline 360 thus offers an effective platform for developing virtual laboratory modules which may be widely adapted to suit the specific needs of a variety of laboratory courses.

GMF as an Actin Network Remodeling Factor.

Glia maturation factor (GMF) has recently been established as a regulator of the actin cytoskeleton with a unique role in remodeling actin network architecture. Conserved from yeast to mammals, GMF is one of five members of the ADF-H family of actin regulatory proteins, which includes ADF/cofilin, Abp1/Drebrin, Twinfilin, and Coactosin. GMF does not bind actin, but instead binds the Arp2/3 complex with high affinity. Through this association, GMF catalyzes the debranching of actin filament networks and inhibits actin nucleation by Arp2/3 complex. Here, we discuss GMF's emerging role in controlling actin filament spatial organization and dynamics underlying cell motility, endocytosis, and other biological processes. Further, we attempt to reconcile these functions with its earlier characterization as a cell differentiation factor.

A novel role for WAVE1 in controlling actin network growth rate and architecture.

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

GMF promotes leading-edge dynamics and collective cell migration in vivo.

Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. We show that glia-maturation factor (GMF), which is an Arp2/3 complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R(+) cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment.

GMF severs actin-Arp2/3 complex branch junctions by a cofilin-like mechanism.

BACKGROUND: Branched actin filament networks driving cell motility, endocytosis, and intracellular transport are assembled in seconds by the Arp2/3 complex and must be equally rapidly debranched and turned over. One of the only factors known to promote debranching of actin networks is the yeast homolog of glia maturation factor (GMF), which is structurally related to the actin filament-severing protein cofilin. However, the identity of the molecular mechanism underlying debranching and whether this activity extends to mammalian GMF have remained open questions. RESULTS: Using scanning mutagenesis and total internal reflection fluorescence microscopy, we show that GMF depends on two separate surfaces for debranching. One is analogous to the G-actin and F-actin binding site on cofilin, but we show using fluorescence anisotropy and chemical crosslinking that it instead interacts with actin-related proteins in the Arp2/3 complex. The other is analogous to a second F-actin binding site on cofilin, which in GMF appears to contact the first actin subunit in the daughter filament. We further show that GMF binds to the Arp2/3 complex with low nanomolar affinity and promotes the open conformation. Finally, we show that this debranching activity and mechanism are conserved for mammalian GMF. CONCLUSIONS: GMF debranches filaments by a mechanism related to cofilin-mediated severing, but in which GMF has evolved to target molecular junctions between actin-related proteins in the Arp2/3 complex and actin subunits in the daughter filament of the branch. This activity and mechanism are conserved in GMF homologs from evolutionarily distant species.

Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa.

BACKGROUND: Usher syndrome type II (USH2) is an autosomal recessive disorder characterised by retinitis pigmentosa (RP) and mild to moderate sensorineural hearing loss. Mutations in the USH2A gene are the most common cause of USH2 and are also a cause of some forms of RP without hearing loss (ie, non-syndromic RP). The USH2A gene was initially identified as a transcript comprised of 21 exons but subsequently a longer isoform containing 72 exons was identified. METHODS: The 51 exons unique to the long isoform of USH2A were screened for mutations among a core set of 108 patients diagnosed with USH2 and 80 patients with non-syndromic RP who were all included in a previously reported screen of the short isoform of USH2A. For several exons, additional patients were screened. RESULTS: In total, 35 deleterious mutations were identified including 17 nonsense mutations, 9 frameshift mutations, 5 splice-site mutations, and 4 small in-frame deletions or insertions. Twenty-seven mutations were novel. In addition, 65 rare missense changes were identified. A method of classifying the deleterious effect of the missense changes was developed using the summed results of four different mutation assessment algorithms, SIFT, pMUT, PolyPhen, and AGVGD. This system classified 8 of the 65 changes as 'likely deleterious' and 9 as 'possibly deleterious'. CONCLUSION: At least one mutation was identified in 57-63% of USH2 cases and 19-23% of cases of non-syndromic recessive RP (calculated without and including probable/possible deleterious changes) thus supporting that USH2A is the most common known cause of RP in the USA.

Diagnosis in a patient with fundus albipunctatus and atypical fundus changes.

We report a case of an 11-year old Caucasian female with nyctalopia since early childhood with an atypical clinical presentation of fundus albipunctatus (FA), and a novel mutation in the RDH5 gene. In addition to white spots in the fundus, patchy areas of hypopigmentation were noted, which were reminiscent for an early stage of retinitis punctata albescens (RPA). Electroretinographic testing (ERG) showed a non-detectable, dark adapted, isolated rod response and a markedly decreased combined rod and cone response to an achromatic stimulus. After patching one eye overnight, both the isolated rod response and combined rod and cone scotopic white flash response were normal. A Goldmann-Weekers dark adapted final threshold response was also within the normal range. The patient showed a previously reported heterozygous mutation for Gly238Trp, and a novel Arg157Gln mutation. Genetic testing and extended ERG and psychophysical testing may be necessary to diagnose FA from early stages of progressive RPA.

Alleles in the HtrA serine peptidase 1 gene alter the risk of neovascular age-related macular degeneration.

OBJECTIVE: To examine if the genes encoding the pleckstrin homology domain-containing protein gene (PLEKHA1), hypothetical LOC387715/ARMS2 gene, and HtrA serine peptidase 1 gene (HTRA1) located on the long arm of chromosome 10 (10q26 region) confer risk for neovascular age-related macular degeneration (AMD) in an independent or interactive manner when controlling for complement factor H gene (CFH) genotype and smoking exposure. DESIGN: Retrospective matched-pair case-control study. PARTICIPANTS: Hospital clinic-based sample of 134 unrelated patients with neovascular AMD who have a sibling with normal maculae (268 subjects). METHODS: Disease status was ascertained by at least 2 investigators by review of fundus photographs and/or fluorescein angiography according to the Age-Related Eye Disease Study grading scale. If necessary, a home retinal examination was performed (n = 6). A combination of direct sequencing and analysis of 8 highly polymorphic microsatellite markers was used to genotype 33 megabases of the 10q26 region on leukocyte DNA. Smoking history was obtained via a standardized questionnaire and measured in pack-years. The family-based association test, haplotype analysis, multiple conditional logistic regression, and linkage analysis were used to determine significant associations. MAIN OUTCOME MEASURE: Neovascular AMD status. RESULTS: Of the 23 variants we identified in the 10q26 region, 6 were significant. Four of the 6 were novel and included 2 genotypes that reduced risk of AMD. Many single-nucleotide polymorphisms (SNPs), including the previously reported variants rs10490924 (hypothetical LOC387715/ARMS2) and rs11200638 (HTRA1), defined 2 significant haplotypes associated with increased risk of neovascular AMD. The coding HTRA1 SNP rs2293870, not part of the significant haplotypes containing rs10490924 and rs11200638, showed as strong an association with increased susceptibility to neovascular AMD. Linkage analysis supported our findings of SNP association (P<10(-15)). No significant interactions were found between any of the SNPs in the 10q26 and smoking or between these SNPs and CFH genotype. CONCLUSIONS: Independent of CFH genotype or smoking history, an individual's risk of AMD could be increased or decreased, depending on their genotype or haplotype in the 10q26 region.

Low prevalence of lecithin retinol acyltransferase mutations in patients with Leber congenital amaurosis and autosomal recessive retinitis pigmentosa.

PURPOSE: To determine the the prevalence of pathogenic mutations in the gene encoding lecithin retinol acyltransferase (LRAT) in patients from North America with either Leber congenital amaurosis (LCA) or autosomal recessive retinitis pigmentosa (ARRP). METHODS: Exon 1, exon 2, and the coding region of exon 3 of LRAT were PCR-amplified and directly sequenced from the leukocyte DNA of 82 unrelated patients with LCA and 190 unrelated patients with ARRP. RESULTS: One isocoding change was found in this screen of LRAT (Glu114 GAG>GAA; c.342), and 5 other sequence changes were found in intronic or untranslated regions of the gene. None of these changes were predicted to affect the encoded protein and were therefore deemed non-pathogenic. CONCLUSIONS: LRAT mutations are likely a rare cause of LCA among patients from North America.