Biology subjects

Quintero-Carmona, O. A.

Publications and source records attributed to Quintero-Carmona, O. A..

5 recordsLinked to original sources

bioRxiv2026

Exploring phosphoregulation of MYO3A using quantitative fluorescence image analysis in COS7 cells

Myosin 3A (MYO3A) is an unconventional myosin involved in the formation and maintenance of hair-cell stereocilia of the sensory epithelia in the inner ear. The kinase domain has been implicated in phosphoregulation of MYO3A activity through intermolecular autophosphorylation. Previous studies using mass spectrometry identified two potential phosphorylation sites in the motor domain. To investigate the regulatory roles of these sites, we generated glutamic acid point mutations in our mchr-MYO3A{Delta}K construct to mimic phosphorylation and assayed the constructs for their ability to tip-localize and influence filopodial density via transfection into COS7 cells. The phosphomimic constructs were less able to generate filopodia when compared to wildtype constructs. To gain a better understanding of the phosphoregulation of MYO3A, we transfected COS7 cells with mchr-MYO3A{Delta}K in combination with GFP-tagged full-length MYO3A (GFP-MYO3AFL), or GFP attached to just the kinase domain of MYO3A (GFP-MYO3AKIN). Coexpression of mchr-MYO3A{Delta}K with either construct resulted in decreased mchr-MYO3A levels at the tips of filopodia and fewer filopodia at the edge of the cell, compared to cells expressing mchr-MYO3A{Delta}K alone. This implies that the kinase domain does not require motor activity to contribute to phosphoregulation of MYO3A, and that MYO3A phosphoregulation may be influencing filopodia initiation. Informatic analyses and structural predictions suggest that the two phosphorylation sites in the motor domain inhibit actin/MYO3A interactions. Taken together, these analyses link MYO3A phosphorylation with the regulation of its ability to create actin protrusions such as filopodia and stereocilia.

cell biology↗

bioRxiv2025

Cells stably expressing shRNA against MYO10 display altered cell motility

Myosin-X (MYO10) is an actin-based motor protein involved in cytoskeletal dynamics, membrane interactions, and integrin-mediated adhesion. To investigate MYO10s cellular roles, we generated MYO10 knockdown (MYO10KD) HeLa and COS7 cell lines using lentiviral shRNA. Compared to wild-type cells, both MYO10KD lines showed reduced proliferation and impaired cell migration in wound assays. There were fewer edge filopodia in HeLa cells. Additionally, MYO10KD cells demonstrated increased spreading on laminin-coated substrates, suggesting altered integrin activation and cytoskeletal linkage. Our results reinforce MYO10s importance in cell proliferation, adhesion, and migration; these MYO10KD lines provide an accessible cell culture model for further study of MYO10.

cell biology↗

bioRxiv2025

Transcriptomic Analysis of CAD Cell Differentiation

CAD cells were derived from Cath.a cells, a mouse central nervous system catecholaminergic cell line. Serum-starved CAD cells undergo morphological changes and resemble isolated neurons when observed by microscopy. We carried out an RNAseq transcriptomic analysis to examine differentiated CAD cells for expression signatures related to neuronal functions, identifying [~]1900 transcripts whose expression changed with differentiation. Pathview analysis identified [~]80 KEGG pathway gene sets that were differentially expressed, including upregulation of at least 13 neuron-related pathways. This dataset can be explored more deeply, allowing further investigation into expression changes relevant to studying neuronal functions in this easy-to-culture model system.

neuroscience↗

bioRxiv2024

INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement

Proper regulation of organelle dynamics is critical for cellular function, but the mechanisms coordinating multiple organelles remain poorly understood. Here we show that actin polymerization mediated by the endoplasmic reticulum (ER)-anchored formin INF2 acts as a master regulator of organelle morphology and movement. Using high-resolution imaging, we demonstrate that INF2-polymerized actin filaments assemble at ER contact sites on mitochondria, endosomes, and lysosomes just prior to their fission. Genetic manipulation of INF2 activity alters the size, shape and motility of all three organelles. Our findings reveal a conserved mechanism by which the ER uses actin polymerization to control diverse organelles, with implications for understanding organelle dysfunction in neurodegenerative and other diseases. This work establishes INF2-mediated actin assembly as a central coordinator of organelle dynamics and inter-organelle communication.

cell biology↗

bioRxiv2021

TRAK proteins encode distinct MIRO-dependent and MIRO-independent mechanisms for associating with the mitochondrial outer membrane

Current models suggest that MIRO GTPases anchor cytoskeletal motors to the mitochondrial outer membrane (MOM). However, our previous findings indicate that the unconventional myosin, MYO19, interacts with MIRO weakly and that a MIRO-independent MOM-localizing domain interacts more tightly with the MOM. To test the hypothesis that other MIRO interactors may also have MIRO-independent MOM-binding, we examined interactions between TRAK proteins (microtubule motor-mitochondria adaptor proteins) and the MOM via quantitative fluorescence microscopy and steady-state kinetic approaches. Using GFP-TRAK truncations expressed in MIRO1-2 double knockout mouse embryonic fibroblasts, we identified a MIRO-independent mitochondrial binding domain in the C-terminus of TRAK1 and TRAK2, sufficient for MOM-localization similar to what we observed for full length GFP-TRAK proteins. The MIRO-binding domains (MBD) of the TRAK proteins were only able to localize to mitochondria in the presence of ectopic expression of MIRO. Importantly, fluorescence recovery after photobleaching (FRAP) demonstrated that the steady-state kinetics of TRAKMBD/MIRO interactions were faster-exchanging than for either full-length TRAK or the TRAK C-terminal MOM-binding domain expressed alone. These data support a model where faster-exchanging TRAK/MIRO associations could support initial association and/or TRAK activation, while MIRO-independent binding contributes significantly to tighter association to the MOM.

cell biology↗