Biology subjects

Vasquez, C. G.

Publications and source records attributed to Vasquez, C. G..

3 recordsLinked to original sources

bioRxiv2026

Comparison of Techniques for Isolating Small Extracellular Vesicles from Drosophila Larval Hemolymph

Extracellular vesicles (EVs) are membrane-delimited nanoparticles, secreted by virtually all tested cell types to mediate intercellular and interorgan communication by transporting biomolecules, such as proteins, nucleic acids, and lipids, to recipient cells. Many EV isolation techniques have been developed for mammalian EVs and exploit specific EV properties, such as size, density, and solubility. Although Drosophila has emerged as a simple yet robust animal model for studying fundamental EV biology in its native context, it remains unclear whether commonly used EV isolation techniques can be applied to hemolymph (Drosophila blood). In this study, we first provide an in-depth characterization of particles in hemolymph using two complementary particle analysis techniques: dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). We then evaluate the performance of three commonly used small EV isolation methods--solvent precipitation, ultracentrifugation, and size-exclusion chromatography--for their ability to isolate small EVs from Drosophila larval hemolymph. All three methods can enrich small EVs from hemolymph to varying degrees, but none completely remove circulating proteins and lipoproteins. In particular, size-exclusion chromatography yields the purest small EV fractions, as evidenced by the enrichment of Drosophila orthologs of human small EV markers, including Tetraspanin 42Ee (Tsp42Ee), Tetraspanin 42Ed (Tsp42Ed), Tetraspanin 96F (Tsp96F), and Annexin B11 (AnxB11), although it produces the lowest protein yield. Altogether, our study provides practical guidance on both selecting an appropriate small EV isolation method and rigorously characterizing isolated small EV fractions according to specific research needs. More broadly, the knowledge gained from this study provides a framework for the isolation and characterization of small EVs in other insects, thereby facilitating future EV research across diverse insect species.

cell biology↗

bioRxiv2020

Lattice micropatterning of electron microscopy grids for improved cellular cryo-electron tomography throughput

Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environment. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and the thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

bioengineering↗

bioRxiv2019

A geometry-based model describes lumen stability in epithelial cells

A continuous sheet of epithelial cells surrounding a hollow opening, or lumen, defines the basic topology of numerous organs. De novo lumen formation is a central feature of embryonic development whose dysregulation leads to congenital and acquired diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by recent experiments in the mouse blastocyst. High luminal pressure should produce lumen surfaces that bow outwards toward the surrounding cells. However, lumens formed in other embryonic tissues adopt highly irregular shapes, with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen growth in all cases. We used three-dimensional live-cell imaging to study the physical mechanism of lumen formation in Madin Darby Canine Kidney (MDCK) cell spheroids, a canonical cell-culture model for lumenogenesis. Our experiments revealed that neither lumen pressure nor the actomyosin cytoskeleton were required to maintain lumen shape or stability. Instead, we find that, in our model system, lumen shape results from simple geometrical factors tied to the establishment of apico-basal polarity. A quantitative physical model that incorporates cell geometry, cortical tension, and intraluminal pressure can account for our observations as well as cases in which pressure indeed plays a dominant role. Our results thus support a unifying physical mechanism for the formation of luminal openings in a variety of physiological contexts.

biophysics↗