Biology subjects

Perez, G. I.

Publications and source records attributed to Perez, G. I..

5 recordsLinked to original sources

bioRxiv2025

The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

DNA double strand breaks (DSBs) are widely considered the most cytotoxic DNA lesions occurring in cells because they physically disrupt the connectivity of the DNA double helix. Homologous recombination (HR) is a high-fidelity DSB repair pathway that copies the sequence spanning the DNA break from a homologous template, most commonly the sister chromatid. How both DNA ends, and the sister chromatid are held in close proximity during HR is unknown. Here we demonstrate that the PST repeat region of MDC1 is a mutlivalent nucleosome binding domain, sufficient to tether chromatin in multiple contexts. In mitotic cells the affinity of the PST repeats for chromatin is downregulated by phosphorylation to prevent chromosome missegregation, while still contributing to DNA break tethering by the MDC1-TOPBP1-CIP2A complex. In interphase, the PST repeat region is critical for RAD51 focus formation but not the recruitment of 53BP1 to DNA breaks, consistent with a chromatin tethering function. In total, this work demonstrates that the PST repeat region of MDC1 is a multivalent chromatin binding domain with tunable affinity that contributes to DNA break tethering during HR and in mitosis.

cell biology↗

bioRxiv2023

AMPK Regulates Phagophore-to-Autophagosome Maturation

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

cell biology↗

bioRxiv2022

Phosphatidylserine-exposing medium/large extracellular vesicles: potential cancer biomarkers

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) also externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

cancer biology↗

bioRxiv2021

In vitro and in vivo analysis of microvesicle-mediated metastasis using a bright, red-shifted bioluminescent reporter protein of extracellular vesicles

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. Our study focused on a novel method to image EV subtypes and their biodistribution in vivo. Regardless of injection routes, we established that reporter EVs isolated from murine mammary carcinoma cells expressing PalmReNL, which utilizes bioluminescence resonance energy transfer (BRET), localized to the lungs. This new EV reporter allowed highly sensitive EV tracking in vitro and in vivo and enabled us to begin studies to understand the commonalities and functional differences of the EV subtypes. We demonstrated the early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple injections of the microvesicle (MV)-enriched fraction derived from mammary carcinoma cells. In addition, the results we present here show that tumor cell-derived MVs act on distant tissues through upregulating LC3 expression within the lung.

cancer biology↗

bioRxiv2021

TCAB1 is necessary for telomerase assembly

Localization of a wide variety of RNAs to non-membrane bound cellular compartments such as nucleoli, Cajal bodies, and stress-granules is critical for their function and stability. The molecular mechanisms that underly the recruitment and exclusion of specific RNAs from these phase-separated organelles is incompletely understood. Telomerase is a ribonucleoprotein (RNP) that is composed of the reverse transcriptase protein TERT, the telomerase RNA (TR), and several auxiliary proteins that associate with TR, including TCAB1. Here we show that in the absence of TCAB1, TR is tightly bound to the nucleolus, while TERT localizes to the nucleoplasm and is largely excluded from the nucleolus, significantly reducing telomerase assembly. Thus, nuclear compartmentalization by the non-membrane bound nucleolus counteracts telomerase assembly and TCAB1 is required to exclude the telomerase RNA from the nucleolus. Our work provides insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase separation proposes a new model explaining the critical role of TCAB1 in telomerase function.

cell biology↗