Biology subjects

Morales, E. A.

Publications and source records attributed to Morales, E. A..

5 recordsLinked to original sources

bioRxiv2026

WHaloForce enables chemigenetic imaging of molecular tension in living cells and animals

Piconewton forces borne by individual proteins within complex assemblies underlie cell adhesion, migration, and tissue morphogenesis, yet remain difficult to image in living systems. Here, we introduce WHaloForce, a chemigenetic HaloTag-based tension sensor that converts force-dependent relief of tryptophan-mediated dye quenching into a fluorescence lifetime change. Optical tweezers revealed a switch-like unquenching transition near 5 pN, and the sensor responded reversibly to force changes in cells. WHaloForce enabled quantitative tension imaging of diverse force-bearing proteins (vinculin, E-cadherin, -catenin, and laminin) in mammalian cells, mouse tissue, and C. elegans. Bright synthetic dyes made tension measurements possible at endogenous expression levels. In C. elegans, vinculin and laminin showed opposite tension patterns between tissues, revealing distinct force-transmission routes through adhesions and the extracellular matrix. During ovulation, laminin tension accumulated over repeated stretch-relaxation cycles, scaling with cumulative loading history. WHaloForce thus offers a modular platform for imaging spatiotemporal tension patterns in living systems.

cell biology↗

bioRxiv2026

Adapting Upright Light Sheet Fluorescence Microscopy for Imaging at Air-Liquid Interface

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

bioengineering↗

bioRxiv2024

Restricting CAR T Cell Trafficking Expands Targetable Antigen Space

Chimeric antigen receptor (CAR) T cells are an effective treatment for some blood cancers. However, the lack of tumor-specific surface antigens limits their wider use. We identified a set of surface antigens that are limited in their expression to cancer and the central nervous system (CNS). We developed CAR T cells against one of these antigens, LINGO1, which is widely expressed in Ewing sarcoma (ES). To prevent CNS targeting, we engineered LINGO1 CAR T cells lacking integrin 4 (A4ko), an adhesion molecule essential for migration across the blood-brain barrier. A4ko LINGO1 CAR T cells were efficiently excluded from the CNS but retained efficacy against ES. We show that altering adhesion behavior expands the set of surface antigens targetable by CAR T cells. One sentence summaryAltering integrin-mediated adhesion provides tumor selectivity to CAR T cells by preventing homing to defined normal tissues but retaining tumor trafficking and anti-tumor activity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/579002v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@256b53org.highwire.dtl.DTLVardef@a94775org.highwire.dtl.DTLVardef@1926f62org.highwire.dtl.DTLVardef@cec52e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Mitotic spindle positioning protein (MISP) is an actin bundler that senses ADP-actin and binds near the pointed ends of filaments

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. Using in vitro TIRF microscopy assays, we found that MISP exhibits a clear binding preference for filaments enriched in ADP-actin monomers. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

cell biology↗

bioRxiv2021

Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli

Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the rootlet end of the microvillus. MISP promotes rootlet elongation in cells, and purified MISP exhibits potent filament bundling activity in vitro. MISP-bundled filaments also recruit fimbrin, which further elongates and stabilizes bundles. MISP confinement to the rootlet is enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. These discoveries reveal how epithelial cells optimize apical membrane surface area and offer insight on the remarkable robustness of microvilli biogenesis.

cell biology↗