Study Of Protein Trafficking Research Articles

Platinum Nanozyme Probes for Cellular Imaging by Electron Microscopy

The highly efficient peroxidase‐like activity of platinum nanozymes (3–20 nm size) is exploited within the complex cellular environment to catalyze the oxidation of the DAB substrate, producing an electron‐dense signal around the nanozyme surface, upon osmium staining. It is proved that such nanozyme amplification can achieve a catalytic signal enhancement up to 10‐fold, enabling the quick detection of the Pt particles (even of 3 nm size) by transmission electron microscopy (TEM) also at low magnification and across wide fields of view in the intricate intracellular milieu. The developed procedure is ideally suited to overcome standard amplification strategies currently used in TEM analysis, such as gold or silver enhancements. Furthermore, the wide versatility of the Pt‐nanozyme probes in TEM imaging is demonstrated in immuno‐EM and protein trafficking studies, showing their potential to track the subcellular localization of target biomolecules at both low and high magnifications. These results suggest that the use of nanozymes might represent a paradigm shift in the conventional amplification systems currently employed in electron microscopy for cellular analyses, offering enhanced imaging capabilities.

Dynamic tandem proximity-based proteomics-Protein trafficking at the proteome-scale.

TransitID is a new methodology based on proximity labeling allowing for the study of protein trafficking a the proteome scale.

Fluorogen‐Activating Proteins as a powerful new imaging tool for quantitative protein trafficking studies in yeast

Recent advantages of genetically encoded fluorescent probes have led to the development of fluorogen-activating proteins (FAPs). This technology has two components: a non-fluorescent single chain antibody (SCA) that can be fused to a protein of interest and fluorogens, which in our case are derivatives of malachite green that are non-fluorescent when free in solution. When the SCA and fluorogen bind, there is a 20,000-fold fluorescent increase relative to unbound dye. The FAP-technology has two major advantages to standard fluorescent probes: i) the fluorogen exists as either a membrane-permeant or impermeant form and this allows for selective labeling of cell surface proteins and (2) since SCA does not fluoresce when it is not bound by dye and so there is no background fluorescence during the imaging of other markers until after the fluorogen is added. Although developed in yeast, this technology surprisingly has not been applied in this model system until our recent studies. In order to make this technology more available to the yeast community, we have optimized the SCA sequence for yeast expression, created a series for FAP-tagged plasmid constructs, and generated a suite of intracellular localization markers to aid in cell biology studies in yeast. We further employ the FAP technology to study the trafficking of the G-protein coupled receptor Ste3 and its regulation by the alpha-arrestins, an important class of trafficking adaptor.

Fluorogenic Probing of Membrane Protein Trafficking.

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

Structural basis for the geometry-driven localization of a small protein

In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM(P9A)) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.

Detailed Topology Mapping Reveals Substantial Exposure of the “Cytoplasmic” C-Terminal Tail (CTT) Sequences in HIV-1 Env Proteins at the Cell Surface

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20–50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

Photoswitchable Biocompatible Polymer Dots Doped with Diarylethene

Molecular photoswitches can be employed for the study of protein trafficking in living cells and applications in optical memories. Especially, to switch fluorescence, fluorescence quenching mechanism via energy or electron transfer is one of the most fundamental pathways to realize the system of photoswitching. In order to achieve fluorescence photoswitching, photochromic compounds such as diarylethene have been used to toggle fluorescence on and off. For example, photochromic diaryethene induces absorption changes upon light irradiations via cyclization reaction, which would trigger the fluorescence toggling. On the other hand, polymer dots (P-dots) is one of the promising fluorescent probes for the biological applications. We assumed that doping diarylethene into P-dots would realize fabrication of photoswhitchable P-dots via energy transfer mechanism between fluorescent polymer and diarylethene. In this study, we synthesized photoswitchable P-dots doped with diarylethene to toggle the fluorescence back and forth via energy transfer mechanism. We also tried to apply synthesized photoswitchable P-dots toward biological imaging. First, we examined the photoswitching properties with absorption and fluorescence measurements. Fluorescence of P-dots was dramatically quenched upon photoirradiation with UV light and recovered after visible light irradiation. Those photoswitching processes were reversible and could go through at least 5 cycles. We are now applying photoswitchable P-dots synthesized as mentioned above to biological imaging. Details will be discussed at the meeting.

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

Paradigm of Genetic Mosaicism and Lone Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia observed in otherwise healthy individuals. Most lone AF cases are nonfamilial, leading to the assumption that a primary genetic origin is unlikely. In this study, we provide data supporting a novel paradigm that atrial tissue-specific genetic defects may be associated with sporadic cases of lone AF. We sequenced the entire coding region of the connexin 43 (Cx43) gene (GJA1) from atrial tissue and lymphocytes of 10 unrelated subjects with nonfamilial, lone AF who had undergone surgical pulmonary vein isolation. In the atrial tissue of 1 patient, we identified a novel frameshift mutation caused by a single nucleotide deletion (c.932delC) that predicted 36 aberrant amino acids followed by a premature stop codon, leading to truncation of the C-terminal domain of Cx43. The mutation was absent from the lymphocyte DNA of the patient, indicating genetic mosaicism. Protein trafficking studies demonstrated intracellular retention of the mutant protein and a dominant-negative effect on gap junction formation of both wild-type Cx43 and Cx40. Electrophysiological studies revealed no electrical coupling of cells expressing the mutant protein alone and significant reductions in coupling when coexpressed with wild-type connexins. This study reports atrial tissue genetic mosaicism of a novel loss-of-function Cx43 mutation associated with lone AF. These findings implicate somatic genetic defects of Cx43 as a potential cause of AF and support the paradigm that sporadic, nonfamilial cases of lone AF may arise from genetic mosaicism that creates heterogeneous coupling patterns, predisposing the tissue to reentrant arrhythmias.

Processing of Surfactant Protein C Requires a Type II Transmembrane Topology Directed by Juxtamembrane Positively Charged Residues

Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism

AbstractDefects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Membrane traffic in sphingolipid storage diseases.

In this review, we summarize our studies of membrane lipid transport in sphingolipid storage disease (SLSD) fibroblasts. We recently showed that several fluorescent SL analogs were internalized from the plasma membrane predominantly to the Golgi complex of normal cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (The Lancet 1999;354: 901-905). Additional studies showed that cholesterol homeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol (Nature Cell Biol 1999;1: 386-388). Based on these findings, we hypothesize that endogenous sphingolipids, which accumulate in SLSD cells due to primary defects in lipid catabolism, result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. The importance of SL/cholesterol interactions and potential mechanisms underlying the regulation of lipid transport and targeting are also discussed. These studies suggest a new paradigm for regulation of membrane lipid traffic along the endocytic pathway and could have important implications for future studies of protein trafficking as well as lipid transport. This work may also lead to important future clinical developments (e.g. screening tests for SLSD, new methodology for screening drugs which abrogate lipid storage, and possible therapeutic approaches to SLSD).

Invertase fusion proteins for analysis of protein trafficking in yeast

Publisher Summary This chapter describes the assays to measure invertase enzymatic activity, focusing on the secretion of invertase fusion proteins as a reporter in studies of vacuolar protein trafficking. As a reporter protein, invertase enzymatic activity can be measured by both quantitative and qualitative assays. In addition, the invertase protein can be followed by methods that measure the extent and types of glycosylation it receives. Several important features of invertase have contributed to its utility in protein trafficking studies. Its enzymatic activity is not altered when fused to heterologous proteins by recombinant DNA techniques, and because invertase secretion is not essential under standard growth conditions, it can be easily manipulated without affecting cell viability. In addition, other than the N-terminal signal sequence that is cleaved after its translocation into the endoplasmic reticulum (ER), invertase does not appear to contain any sequence information that is essential for its transport through the secretory pathway. Thus, invertase enzymatic activity is accurately localized within the cellular compartment(s) to which the native protein under study is transported.

Recombinant adenovirus is an appropriate vector for endocytotic protein trafficking studies in cultured neurons

Endocytosis of full-length β-amyloid precursor protein (APP) from the plasma membrane contributes to β-amyloid peptide (Aβ) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer’s disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30–35%) at low viral titer (10–20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10–20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.

Preparation of the fission yeast Schizosaccharomyces pombe for ultrastructural and immunocytochemical study.

The fission yeast Schizosaccharomyces pombe serves as a model system for investigating a wide variety of problems in eukaryotic cellular and molecular biology. Although probably most widely studied in relation to the control of the eukaryotic cell cycle () and the events of cell division (,), S. pombe has also received significant attention in studies of protein trafficking and secretion (), cell wall biosynthesis (), cellular and molecular aspects of meiosis, genetic recombination, and spore formation (), signal transduction pathways and control of gene expression (-) and cell growth (,), cell morphology (,) and the cytoskeleton (,). In many ways we can think of S. pombe as essentially a typical eukaryote trapped inside a cell wall, and the resulting constraints on cell morphology confer on it a stereotyped cellular architecture, well suited to ultrastructural analysis. The details of this architecture at both the light- and electron-microscopic level can be found in reference (); here we will only highlight certain features.

Renal epithelial cell polarity.

Recent cell biologic studies of protein trafficking, sorting, and distribution in polarized renal epithelial cells have begun to provide important new insights into the mechanisms involved in generating and maintaining cell surface polarity. Advances in this field have been rapid in the last year, due in part to the development of new approaches to analyzing protein delivery and distribution in polarized renal cells grown in vitro. Sorting signals within apical and basal-lateral membrane proteins have been described that may be involved in the segregation of proteins into different populations of transport vesicles in the trans-Golgi network; the nature of these signals has provided insight into the mechanisms involved. Elements of the cytoskeleton appear to be involved in the delivery of these transport vesicles to the appropriate membrane domain (microtubules) and in the retention of specific proteins in the correct membrane domain (membrane skeleton). Finally, detailed analysis of two prominent renal diseases, ischemia and polycystic kidney disease, indicates that abnormalities in the regulation of membrane protein distribution may be a contributing factor in generating the disease state.