Biology subjects

Gunning, P.

Publications and source records attributed to Gunning, P..

4 recordsLinked to original sources

bioRxiv2026

Intravital single-molecule imaging reveals cytoskeletal turnover as a driver of membrane remodeling in live animals

Understanding how cells regulate plasma membrane architecture inside intact living organs in a live animal has been limited by the inability to directly measure molecular dynamics in vivo. Here we introduce intravital single-molecule microscopy (iSiMM), an imaging approach that enables tracking of individual, endogenously expressed cytoskeletal components at the plasma membrane in live mice. Applying iSiMM to murine acinar secretory cells, we identify discrete basolateral membrane domains built on deeply folded membrane infolds that function as a pre-existing membrane reservoir. Single-molecule measurements reveal continuous, regulated molecular turnover within these domains. Physiological stimulation accelerates cytoskeletal exchange promoting rapid membrane unfolding and cell expansion. Together, these findings establish iSiMM as a general strategy for probing molecular kinetics underlying dynamic cellular behaviors in intact organs. One-sentence summaryIntravital single-molecule microscopy enables direct measurement of molecular kinetics underlying dynamic cellular behaviors in intact living organs.

cell biology↗

bioRxiv2023

Spatial and Temporal Coordination of Force-generating Actin-based Modules Drives Membrane Remodeling In Vivo

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that: a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane; and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

cell biology↗

bioRxiv2023

Small molecule STAT3/5 inhibitors exhibit therapeutic potential in acute myeloid leukemia and extra-nodal natural killer/T cell lymphoma

The oncogenic transcription factors STAT3, STAT5A and STAT5B are essential to steer hematopoiesis and immunity, but their enhanced expression and activation drives the development or progression of blood cancers. Current therapeutic strategies focus on blocking upstream tyrosine kinases, but frequently occurring resistance often leads to disease relapse, emphasizing the need for more targeted therapies. Here we evaluate JPX-0700 and JPX-0750, which are STAT3/5-specific covalent cysteine binders that lead to growth arrest of acute myeloid leukemia (AML) and natural killer/T cell lymphoma (NKCL) cell lines in vitro and in vivo, as well as reduce cell viability of primary AML blasts ex vivo. Our non-PROTAC small molecular weight degraders selectively reduce STAT3/5 activation and total protein levels, as well as downstream target oncogene expression, exhibiting nanomolar to low micromolar efficacy. We found that both AML and NKCL cells hijack STAT3/5 signaling through either upstream activating mutations in tyrosine kinases, activating gain-of-function mutations in STAT3, mutational loss of negative STAT regulators, or genetic gains in anti-apoptotic, pro-proliferative or epigenetic-modifying STAT3/5 targets. Moreover, we have shown synergistic inhibitory action of JPX-0700 and JPX-0750 upon combinatorial use with approved chemotherapeutics (doxorubicin, daunorubicin, cytarabine), epigenetic enzyme blocker vorinostat, tyrosine kinase inhibitor cabozantinib or BCL-2 inhibitor venetoclax. Importantly, JPX-0700 or JPX-0750 treatment reduced leukemic cell growth in human AML/NKCL xenograft mouse models without adverse side effects. These potent small molecule degraders of STAT3/5 could propel further clinical development for use in AML and NKCL patients.

cancer biology↗

bioRxiv2020

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

cell biology↗