A MATLAB-based Instrument Control (MIC) package for fluorescence imaging.

MATLAB Instrument Control (MIC) is a software package designed to facilitate data collection for custom-built microscopes. Utilizing object-oriented programming, MIC provides a class for each low-level instrument. These classes inherit from a common MIC abstract class, ensuring a uniform interface across different instruments.

Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions.

Background: Actin stress fibers (SFs) and focal adhesions (FAs) are dynamic structures crucial to a range of cell behaviors including cell morphology, cell migration, proliferation, survival, and differentiation. The Arf GAP ASAP1 affects both SFs and FAs. Here, we test the hypothesis that two domains with distinct biochemical activities in ASAP1, the BAR domain that binds actin and nonmuscle myosin 2 (NM2) and the Arf GAP domain, which is necessary for inducing hydrolysis of GTP bound to Arf, coordinately regulate the structures.

CD28 shapes T cell receptor signaling by regulating Lck dynamics and ZAP70 activation.

Introduction: T cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response.

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation.

Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs.

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains.

mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners.

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners.

The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types.

SMITE: Single Molecule Imaging Toolbox Extraordinaire (MATLAB).

Fluorescence single molecule imaging comprises a variety of techniques that involve detecting individual fluorescent molecules. Many of these techniques involve localizing individual fluorescent molecules with precisions below the diffraction limit, which limits the spatial resolution of (visible) light-based microscopes. These methodologies are widely used to image biological structures at the nanometer scale by fluorescently tagging the structures of interest, elucidating details of the biological behavior observed.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling.

Endolysosomal Mesoporous Silica Nanoparticle Trafficking along Microtubular Highways.

This study examines intra- and intercellular trafficking of mesoporous silica nanoparticles along microtubular highways, with an emphasis on intercellular bridges connecting interphase and telophase cells. The study of nanoparticle trafficking within and between cells during all phases of the cell cycle is relevant to payload destination and dilution, and impacts delivery of therapeutic or diagnostic agents. Super-resolution stochastic optical reconstruction and sub-airy unit image acquisition, the latter combined with Huygens deconvolution microscopy, enable single nanoparticle and microtubule resolution.

Robust, fiducial-free drift correction for super-resolution imaging.

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS.

ATG9A protects the plasma membrane from programmed and incidental permeabilization.

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood.

MERIT, a cellular system coordinating lysosomal repair, removal and replacement.

Unlabelled: Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal mbrane par, removal and replacemen.

Galectin-3 Coordinates a Cellular System for Lysosomal Repair and Removal.

Endomembrane damage elicits homeostatic responses including ESCRT-dependent membrane repair and autophagic removal of damaged organelles. Previous studies have suggested that these systems may act separately. Here, we show that galectin-3 (Gal3), a β-galactoside-binding cytosolic lectin, unifies and coordinates ESCRT and autophagy responses to lysosomal damage.

γ-secretase inhibitor DAPT mitigates cisplatin-induced acute kidney injury by suppressing Notch1 signaling.

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin-induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells.

Sequential super-resolution imaging using DNA strand displacement.

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence.

Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics.

Dysferlin forms a dimer mediated by the C2 domains and the transmembrane domain in vitro and in living cells.

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice.

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown.