Biology subjects

Albanesi, J. P.

Publications and source records attributed to Albanesi, J. P..

3 recordsLinked to original sources

bioRxiv2021

A mutation associated with Charcot-Marie-Tooth disease enhances the formation of stable dynamin 2 complexes in cells

Mutations in dynamin 2 (DNM2) have been associated with two distinct motor disorders, Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). The majority of these mutations are clustered in the pleckstrin homology domain (PHD) which engage in intramolecular interactions that suppress dynamin self-assembly and GTPase activation. CNM mutations in the PHD interferes with these intramolecular interactions, thereby blocking the formation of the auto-inhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for lipid PIP2 binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations cause either one disease or the other, despite their close proximity within the PHD sequence. We show that at least one CMT-causing mutant, lacking residues 555DEE557 ({Delta}DEE), displays this inability to undergo auto-inhibition as observed in CNM-linked mutants. This {Delta}DEE deletion mutant induces the formation of abnormally large cytoplasmic inclusions similar to those observed for CNM-linked mutant R369W. We also found substantially reduced migration from the membrane of the {Delta}DEE deletion mutant. These findings call into question the molecular mechanism currently believed to underlie the absence of pathogenic overlap between DNM2-dependent CMT and CNM.

cell biology↗

bioRxiv2021

Differential mobility and self-association of Arc/Arg3.1 in the cytoplasm and nucleus of living cells

Arc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc in living cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm, and that its movement within PML-NBs is relatively unobstructed.

cell biology↗

bioRxiv2019

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions including cell motility, signal transduction, and virus replication. Here we describe a dual color (DC) extension of the fluorescence z-scan technique which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the co-expression of a second distinctly colored fluorescent protein provides a soluble reference species, which delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2 but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ~16 M and Hill coefficient of n ~2.\n\nSIGNIFICANCEProtein binding to the plasma membrane of cells plays an important role in a multitude of cell functions and disease processes. Quantitative binding studies of protein/membrane interactions are almost exclusively limited to in vitro systems and may produce results that poorly mimic the authentic interactions in living cells. We report quantitative measurements of plasma membrane binding directly in living cells by using dual color z-scan fluorescence, which improves the detection threshold by an order of magnitude compared to our previous single color technique. This advance allowed us to examine the role of mutations on binding affinity and identify the presence of cooperative binding in protein systems with relevance to HIV/AIDS and cancer biology.

biophysics↗