Biology subjects

Gonzales, A.

Publications and source records attributed to Gonzales, A..

5 recordsLinked to original sources

bioRxiv2026

IP3R-TRPM4 Coupling Determines the Spatial Reach of Pericyte-Mediated Capillary Constriction

Ensheathing pericytes extend multiple projections that wrap around capillary vessels to regulate diameter and direct blood flow distribution across the microvascular network. At capillary bifurcations, individual projections wrap around branches of different diameters, positioning the pericyte to simultaneously regulate multiple vessel segments. Using optogenetic tools in acta2-opto-1AR and acta2-CatCh mice, we show that Gq-coupled receptor activation confines contractile responses to the projection receiving the stimulus, whereas direct membrane depolarization propagates to projections wrapping neighboring capillary branches via gap junction-independent mechanisms. Computational modeling revealed that TRPM4, a Ca2+-activated nonselective cation channel, uniquely couples local Ca2+ signals to voltage-gated Ca2+ channel activation in projections on neighboring branches at physiologically relevant channel abundances. Ca2+ imaging identified two kinetically distinct event populations: slow, low-amplitude IP3R-mediated events and fast, high-amplitude VGCC-mediated transients. Sustained low-amplitude signals selectively maintained TRPM4 activation and enabled cross-projection propagation, whereas brief high-amplitude transients drove rapid channel inactivation. Proximity ligation assays confirmed nanoscale colocalization of TRPM4 and IP3 receptors in pericytes. Focal IP3 photolysis produced constriction across projections wrapping multiple capillary branches that was abolished by TRPM4 blockade and enhanced by PKC-mediated augmentation of TRPM4 expression. These findings identify an IP3R-TRPM4 signaling axis as a molecular switch that gates whether capillary constriction remains branch-specific or coordinates across the pericyte, enabling precise stimulus-dependent control of blood flow distribution in the microvasculature.

physiology↗

bioRxiv2025

A zebrafish seizure model of cblX syndrome reveals a dose-dependent refractory response to mTor inhibition.

Mutations in the transcriptional cofactor HCFC1 cause methylmalonic aciduria and homocystinemia, cblX type (cblX) (MIM#309541), non-syndromic X-linked intellectual disability (XLID), and focal epilepsy. Zebrafish studies have revealed increased activation of the Akt/mTor signaling pathway after mutation of hcfc1a, one ortholog of HCFC1. mTOR hyperactivation is linked to seizures and its inhibition alleviates epilepsy in other preclinical models. We hypothesized that mTor overactivity in hcfc1a mutant zebrafish increases seizure susceptibility and/or severity. We employed a two-concentration model of the seizure inducing agent, pentylenetetrazol (PTZ), with or without pretreatment of the mTor inhibitor, torin1. Mutation of hcfc1a did not increase seizure susceptibility at sub-optimal concentrations of PTZ and the pharmaceutical inhibition of mTor reduced seizure severity when utilized at a dose of 250nM. Higher doses of torin1 treatment exacerbated seizure response in mutant larvae but not in wildtype siblings. These data suggest that in an hcfc1a deficient background, moderate inhibition of mTor signaling may partially alleviate seizure phenotypes, however, over-inhibition of the pathway causes a refractory response to PTZ. Collectively, we present a model that can be used to test dose response and for the development of combinatorial treatment approaches in a high throughput manner.

developmental biology↗

bioRxiv2023

Motion Blur Microscopy

Imaging and characterizing the dynamics of cellular adhesion in blood samples is of fundamental importance in understanding biological function. In vitro microscopy methods are widely used for this task, but typically require diluting the blood with a buffer to allow for transmission of light. However whole blood provides crucial mechanical and chemical signaling cues that influence adhesion dynamics, which means that conventional approaches lack the full physiological complexity of living microvasculature. We propose to overcome this challenge by a new in vitro imaging method which we call motion blur microscopy (MBM). By decreasing the source light intensity and increasing the integration time during imaging, flowing cells are blurred, allowing us to identify adhered cells. Combined with an automated analysis using machine learning, we can for the first time reliably image cell interactions in microfluidic channels during whole blood flow. MBM provides a low cost, easy to implement alternative to intravital microscopy, the in vivo approach for studying how the whole blood environment shapes adhesion dynamics. We demonstrate the methods reproducibility and accuracy in two example systems where understanding cell interactions, adhesion, and motility is crucial--sickle red blood cells adhering to laminin, and CAR-T cells adhering to E-selectin. We illustrate the wide range of data types that can be extracted from this approach, including distributions of cell size and eccentricity, adhesion durations, trajectories and velocities of adhered cells moving on a functionalized surface, as well as correlations among these different features at the single cell level. In all cases MBM allows for rapid collection and processing of large data sets, ranging from thousands to hundreds of thousands of individual adhesion events. The method is generalizable to study adhesion mechanisms in a variety of diseases, including cancer, blood disorders, thrombosis, inflammatory and autoimmune diseases, as well as providing rich datasets for theoretical modeling of adhesion dynamics.

biophysics↗

bioRxiv2022

Quantitative analyses of T cell motion in tissue reveals factors driving T cell search in tissues

T cells are required to clear infection, moving first in lymph nodes to interact with antigen bearing dendritic cells leading to activation. T cells then move to sites of infection to find and clear infection. T cell motion plays a role in how quickly a T cell finds its target, from initial naive T cell activation by a dendritic cell to interaction with target cells in infected tissue. To better understand how different tissue environments might affect T cell motility, we compared multiple features of T cell motion including speed, persistence, turning angle, directionality, and confinement of motion from T cells moving in multiple tissues using tracks collected with microscopy from murine tissues. We quantitatively analyzed naive T cell motility within the lymph node and compared motility parameters with activated CD8 T cells moving within the villi of small intestine and lung under different activation conditions. Our motility analysis found that while the speeds and the overall displacement of T cells vary within all tissues analyzed, T cells in all tissues tended to persist at the same speed, particularly if the previous speed is very slow (less than 2 {micro}m/min) or very fast (greater than 8 {micro}m/min) with the exception of T cells in the villi for speeds greater than 10 {micro}m/min. Interestingly, we found that turning angles of T cells in the lung show a marked population of T cells turning at close to 180o, while T cells in lymph nodes and villi do not exhibit this "reversing" movement. Additionally, T cells in the lung showed significantly decreased meandering ratios and increased confinement compared to T cells in lymph nodes and villi. The combination of these differences in motility patterns led to a decrease in the total volume scanned by T cells in lung compared to T cells in lymph node and villi. These results suggest that the tissue environment in which T cells move can impact the type of motility and ultimately, the efficiency of T cell search for target cells within specialized tissues such as the lung.

immunology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗