Biology subjects

Gallo, K.

Publications and source records attributed to Gallo, K..

3 recordsLinked to original sources

bioRxiv2024

TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employed a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. Over 300 proteins were identified, forming different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown solid inclusion at the ER exit sites (ERES). TDP43-ERES coaggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delayed ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients.

cell biology↗

bioRxiv2021

Coincident Fluorescence Burst Analysis of dUTP-Loaded Exosome-Mimetic Nanovesicles

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/463914v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13a85b2org.highwire.dtl.DTLVardef@11f6cd3org.highwire.dtl.DTLVardef@219a73org.highwire.dtl.DTLVardef@232f95_HPS_FORMAT_FIGEXP M_FIG C_FIG The targeting functionality and low immunogenicity of exosomes and exosome-mimetic nanovesicles make them promising as drug-delivery carriers. To tap into this potential, accurate non-destructive methods to load them and characterize their contents are of utmost importance. However, their small size, polydispersity and aggregation in solution make quantitative characterizations of their loading particularly challenging. Here we develop an ad-hoc methodology based on a burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their loading. We apply it to study bioengineered nanovesicles, loaded with dUTP cargo molecules, synthetized from detergent-resistant membranes of animal extracellular vesicles and human red blood cells. For both classes of bioengineered nanovesicles we prove, by means of dual-color fluorescence cross-correlation spectroscopy (FCCS), successful loading. Furthermore, by a dual-color coincident fluorescence burst (DC-CFB) analysis of the experimental data, we retrieve size and loading statistics for both types of nanovesicles. The procedure affords single-vesicle characterizations, which are essential for reliable quantitative studies of loading processes in exosomes and exosome-mimetic nanovesicles, especially in light of the typically high heterogeneity of their populations. Moreover, the method implementation can be easily adapted to the investigation of a variety of combinations of different cargo molecules and biological nanovesicles besides the proof-of-principle demonstrations considered in this study. The results provide a powerful characterization tool, well-suited for the optimization of loading processes of biomimetic nanovesicles and their advanced engineering for therapeutic drug delivery.

biophysics↗

bioRxiv2018

Rho-mediated MRTF and YAP1 activation contributes to BRAF inhibitor resistance in Sox9High/Sox10Low melanoma cells.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that approximately 50-60% of melanoma cell lines with acquired vemurafenib resistance activate the RhoA family signaling pathway. RhoAHigh BRAFi-resistant cells are sensitive to the combination of ROCK inhibitors and vemurafenib. Further, these RhoAHigh cells have >100-fold increase in Sox9 expression and >100-fold decrease in Sox10 expression. Two transcriptional co-activators downstream of RhoA, MRTF and YAP1, are activated in Sox9High/Sox10Low BRAFi-resistant cells. Pharmacological inhibition of these transcriptional mechanisms re-sensitizes the cells to vemurafenib. Analysis of human BRAFi-resistant tumors reveals that many resistant tumors show gene expression signatures consistent with increased RhoA activation or activation of the transcriptional co-activators MRTF and YAP1. A subset of melanoma tumors in the TCGA dataset with low Sox10 expression also have elevated RhoA, MRTF, and YAP1 activation signatures. Taken together, these results support the concept of targeting RhoA-regulated gene transcription pathways as a promising approach for treating or preventing BRAFi-resistance in melanoma.

cancer biology↗