Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein necessary but not sufficient for retrograde directed from synapse to cell body. Dynactin a heteromultimeric protein complex, enriched in neurons, that binds both microtubules and cytoplasmic dynein. To determine whether dynactin required transport, we examined effects anti-dynactin antibodies on organelle extruded axoplasm. Treatment axoplasm with p150 Glued...

Axoplasmic organelles in the giant axon of squid have been shown to move on both actin filaments and microtubules switch between during fast axonal transport. The objectives this investigation were identify specific classes axoplasmic that myosin motors involved. We developed a procedure isolate endoplasmic reticulum (ER) from extruded axoplasm reconstitute its movement vitro. isolated ER vesicles moved exogenous adsorbed coverslips an ATP-dependent manner without addition soluble factors....

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene individuals with fibrosis, DeltaF508, causes retention of DeltaF508-CFTR endoplasmic reticulum and leads to absence CFTR Cl(-) channels apical plasma membrane. Rescue by reduced temperature or chemical means reveals that DeltaF508 reduces half-life Because retains some channel activity, increased expression membrane could serve as a potential therapeutic approach for fibrosis. However, little is...

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl⁻) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that Cl⁻ stimulating CFTR channel trafficking from an intracellular pool to apical plasma membrane. To this end, generated green fluorescent protein (GFP)-CFTR expression vector in GFP was linked N terminus of CFTR....

Abstract The superfamily of myosin proteins found in eukaryotic cells is known to contain at least 18 different classes. Members are classified based on the phylogenetic analysis head domains located amino terminus polypeptide. While relationships provide insights into functional relatedness myosins within and between families, evolutionary history not revealed by such studies. In order establish superfamily, we analyzed representation gene families a range organisms covering taxonomic...

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR channels plasma membrane adjusting endocytosis and recycling. However, mechanisms that regulate recycling airway epithelial cells remain unknown, at least because itineraries these are incompletely understood. In a previous study, we demonstrated undergoes trafficking Rab11a-specific apical endosomes human cells. Myosin...

ABSTRACT Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This occurs at speeds 0.5-1.0 μm/second and majority stain with kinesin antibody. These observations demonstrate, for first time, that (0.5-1.0 μm/second) proteins In addition, our studies suggest can be transported non-membrane bound, nonfilamentous subunits axons, is kinesin-dependent. Microtubule-based might therefore provide...

Abstract We report the design and target validation of chimeric peptide EP45, a novel 45 amino acid monomeric dual agonist that contains sequence motifs present within blood glucose-lowering agent exendin-4 (Ex-4) appetite-suppressing PYY(3–36). In new high-throughput FRET assay provides real-time kinetic information concerning levels cAMP in living cells, EP45 recapitulates action Ex-4 to stimulate production via glucagon-like peptide-1 receptor (GLP-1R), while also recapitulating PYY(3–36)...

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl- channel expressed in the apical plasma membrane fluid-transporting epithelia. Although CFTR rapidly endocytosed from of polarized epithelial cells and efficiently recycled back to membrane, little known about molecular mechanisms regulating endocytosis endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated clathrin-mediated cells. goal this study was...

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band microtubules (MB) and trans-marginal material (TBM). The TBM appeared in whole mounts as rough irregular network thin sections surface-delimiting layer completely enclosing nucleus MB. In cells incubated at 0 degrees C for 30 min or more, MB disappeared but remained. reassembly occurred with rewarming, was inhibited by colchicine. Flattened elliptical morphology retained even when MBs were absent....

ABSTRACT The directionality of the actin-dependent motors on squid axoplasmic organelles was determined using actin filaments assembled barbed ends acrosomal processes. Acrosomal processes were isolated from Limulus polyphemus sperm and incubated in monomeric under conditions that promoted end assembly only. Newly stabilized stained with rhodamine-phalloidin presence at verified by flu-orescence microscopy negative contrast electron microscopy. Axoplasmic dissociated extruded axoplasm...

The structure of 14-protofilament microtubules reassembled from dogfish shark brain tubulin was analyzed by high resolution electron microscopy and optical diffraction. simultaneous imaging the protofilaments near far sides these tubules produces a moiré pattern with period approximately 96 nm. Optical diffraction patterns show that 5-nm spots arise for two tubule are not coincident but lie off equator distance 1/192 nm-1. These data provide evidence in containing 14 protofilaments, tilted...

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A known to associate SVs involved in SV trafficking. We performed experiments determine whether Myo5a required for transport neuronal vesicles. In vitro motility assays axoplasm from squid giant axon showed requirement GTPase Myo5a-dependent vesicle transport. Furthermore, mouse...

Abstract Myosin‐V is a versatile motor involved in short‐range axonal/dendritic transport of vesicles the actin‐rich cortex and synaptic regions nerve cells. It binds to several different kinds neuronal by its globular tail domain but mechanism which it recruited these not known. In this study, we used an vitro motility assay derived from axoplasm squid giant axon study effects on vesicles. We found that fragment myosin‐V inhibited actin‐based vesicle displacing native binding The pulled...

Abstract We present a high‐resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form crossbridges which produce microtubule‐based vesicle transport in squid axoplasm. The were found attached surfaces anterogradely transported presence ATP. These made one three filaments uniform diameter. Each filament measured 5–6 nm width 30–35 length. some had splayed apart by pivoting at their base, thereby assuming “V” shape. spread configuration...

Abstract Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans ‐Golgi network and be involved in Golgi ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates but functions transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously myosin‐dependent vesicle was observed upon activation. In addition, bundles formed moved relative each other at an average...