Biology subjects

Sherwood, D. R.

Publications and source records attributed to Sherwood, D. R..

3 recordsLinked to original sources

bioRxiv2022

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.

developmental biology↗

bioRxiv2021

Hemicentin mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissues can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins decline in levels and are not required for B-LINK maintenance. Instead, we discovered that hemicentin also promotes type IV collagen assembly, which accumulates to high levels during egg-laying and sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM connection maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage. SummaryTissue attachment through linking juxtaposed basement membranes (BMs) is crucial for the structure and function of many organs. Gianakas et al. identify a key role for hemicentin and fibulin-1 in initiating BM-BM attachment and type IV collagen in stabilizing this linkage and allowing it to resist high mechanical loads.

developmental biology↗

bioRxiv2021

A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins localized to BMs. Network analysis and screening in C. elegans and zebrafish identified new BM regulators, including ADAMTS, ROBO, and TGF{beta}. More than 100 BM-network genes associate with human phenotypes and by screening 63,039 genomes from families with rare disorders, we discovered loss-of-function variants in LAMA5, MPZL2, and MATN2, and show they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity and role of BMs in human health.

cell biology↗