Biology subjects

Wysolmerski, B.

Publications and source records attributed to Wysolmerski, B..

4 recordsLinked to original sources

bioRxiv2025

Conformational biosensors delineate endosomal G protein regulation by GPCRs

Many GPCRs trigger a second phase of G protein-coupled signaling from endosomes after initiating signaling from the plasma membrane. This inherently requires receptors to increase the concentration of active-state G proteins on the endosome membrane, but how this is achieved remains incompletely understood. We addressed this question by dissecting the regulation of G protein abundance and activity on endosomes following activation of several Gs-coupled GPCRs-the {beta}2-adrenergic receptor, the VIP-1 receptor, and the adenosine 2b receptor-that are natively co-expressed and differ in their ability to internalize after activation. We first verify GPCR-triggered redistribution of Gs from the plasma membrane to a mixed population of intracellular membranes, including endosomes, that is both reversible after receptor inactivation and triggered irrespective of the ability of the GPCR to internalize. We next show that GPCRs trigger this redistribution process at native expression levels and describe a method, using conformational biosensors, to detect endosomal activation of endogenous Gs. Applying this method, we show that GPCR-mediated production of active-state Gs on endosomes depends on receptor endocytosis, whereas increasing the net amount of Gs on endosomes does not. Our results support a model for Gs regulation on endosomes mediated by two spatially separated receptor coupling events-one at the plasma membrane controlling endosomal Gs abundance and another at endosomes controlling Gs activity. Additionally, our results reveal location-bias in the selectivity of G protein activation on endosomes that is differentially programmed by GPCRs in a receptor-specific manner.

cell biology↗

bioRxiv2020

Dietary supplementation with PUFAs rescues the eggshell defects caused by seipin mutations in C. elegans

SEIPIN, an ER membrane protein, plays critical roles in lipid droplet (LD) formation and lipid storage. Dysfunction of SEIPIN causes a variety of human diseases, including lipodystrophy, neuropathies, and male and female infertility. However, the cellular and molecular mechanisms of SEIPIN in causing these diseases are poorly understood. To address such mechanisms, we investigated the functional roles of R01B10.6 (seip-1), the sole SEIPIN1 ortholog in C. elegans, using CRISPR/Cas9 gene editing, and transcriptional assays. SEIP-1::mScarlet is widely expressed throughout development in C. elegans. Three full gene deletion mutants, generated by CRISPR/Cas9, displayed penetrant embryonic lethality. EM imaging and the visualization of reporter genes revealed that the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell, was defective or missing. Intriguingly, depletion of SEIP-1 revealed a perturbed gene expression pattern for fatty acid biosynthesis enzymes, in agreement with the disrupted permeability barrier formation phenotype of the embryos. Lastly, dietary supplementation of PUFAs rescued the embryonic lethality and defective permeability barrier in the deletion mutants. In sum, our study suggests that SEIP-1 may maternally regulate LD biogenesis and maintain lipid homeostasis to orchestrate the formation of the lipid-rich permeability barrier, which is crucial for eggshell formation and embryogenesis.

cell biology↗

bioRxiv2017

Dynein Pulling Forces On Ruptured Nuclei Counteract Lamin-Mediated Nuclear Envelope Repair Mechanisms In Vivo

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance to in vivo events is not known. Here, we use C. elegans embryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE ruptures in vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct step in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE ruptures in vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.

developmental biology↗

bioRxiv2017

A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among these factors and pathways underlies the importance of safeguarding the genome through multiple means.

genomics↗