Elucidating the Mechanisms Behind Basement Membrane Stretching

Abstract Basement membranes (BMs) are dense extracellular matrices that surround and structurally support tissues. A primary component of BMs is type IV collagen, which forms a cross-linked network that protects BMs and tissues from mechanical stress. Although the key role of type IV collagen in structural support is established, how this

network accommodates BM stretching in mechanically active tissues is unknown. BM stretching is critically important in the cardiovascular system, where vessels must expand and contract to accommodate pulsatile forces from the blood. Consistent with this important role, disruptions in the type IV collagen network can lead to

vascular diseases such as vascular thickening in diabetes, cerebral microbleeds, and hemorrhagic stroke. Despite its significance, our understanding of BM stretching has been limited by a lack of in vivo models to study how BMs dynamically expand. To fill this need, I have established the gonadal BM of the visually and genetically

tractable model organism C. elegans as a new in vivo model for BM stretching. I discovered that the region of the gonadal BM specialized for fertilization/ovulation, the spermathecal BM, is stretched dramatically (~2-fold) during ovulation. Using atomic force microscopy (AFM), I found that there is a stiffness gradient within the gonad

with the spermatheca being the least stiff. Furthermore, 20 BM components have been tagged with mNeonGreen or mRuby using CRISPR/Cas-9, making C. elegans the only animal where all major BM components are endogenously tagged. Using these strains, I found: (1) Increased levels of type IV collagen limit stretching during

ovulation; (2) there are high levels of peroxidasin-1, a protein that negatively regulates type IV collagen cross- linking, in the spermathecal BM; and (3) fibulin, a BM protein thought to maintain type IV collagen, is enriched in the spermatheca. My overall hypothesis is that levels and cross-linking of type IV collagen are precisely

regulated to allow the collagen network to maintain BM/tissue integrity while enabling BM/tissue stretching. In Aim 1, I will use genetic analysis, conditional knockdown, live imaging, and AFM to test the hypothesis that type IV collagen levels play a crucial role in BM stretching (low levels promote decreased

stiffness, increased stretching and eventual rupture; high levels increase stiffness and restrict stretching) and determine whether the effects of type IV collagen α1 and α2 human disease mutations in C. elegans are due to altered levels or location in unique domains associated with BM stretching. Aim 2 will test the hypothesis that

low type IV collagen cross-linking, mediated by peroxidasins, promotes BM stretching by reducing BM stiffness and that fibulin maintains type IV collagen levels in this dynamic environment. In Aim 3, I will determine if additional components regulate BM stretching by conducting a bioinformatics-driven RNAi screen to identify

vascular disease associated genes that affect BM stretching. Ultimately, I expect my work will show that precise type IV collagen levels and cross-linking are critical for balancing tissue support and BM stretching and that these mechanisms are perturbed in human vascular disease, making them ideal targets for more effective therapies.