Visualization of self-delivering hydrophobically modified siRNA cellular internalization.

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs) that efficiently enter cells and tissues without the need for formulation.

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone.

The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein.

Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton.

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss.

A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity.

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown.

Protein kinase Cα and integrin-linked kinase mediate the negative axon guidance effects of Sonic hedgehog.

In addition to its role as a morphogen, Sonic hedgehog (Shh) has also been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. However, the noncanonical signaling pathways that mediate the guidance effects of Shh protein remain poorly understood. We demonstrate that a novel signaling pathway consisting of protein kinase Cα (PKCα) and integrin-linked kinase (ILK) mediates the negative guidance effects of high concentration of Shh on retinal ganglion cell (RGC) axons.

Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands.

Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling.

The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin.

Advances in microscopy techniques.

Context: Advances in microscopy enable visualization of a broad range of new morphologic features.

Objective: To review and illustrate advances in microscopy with relevance to pathologists.

Data Sources: Literature review and new observations.

A novel role for the centrosomal protein, pericentrin, in regulation of insulin secretory vesicle docking in mouse pancreatic beta-cells.

The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown.

MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system.

Chemokine signaling guides regional patterning of the first embryonic artery.

The aorta traverses the body, yet little is known about how it is patterned in different anatomical locations. Here, we show that the aorta develops from genetically distinct endothelial cells originating from diverse locations within the embryo. Furthermore, chemokine (C-X-C motif) receptor 4a (cxcr4a) is restricted to endothelial cells derived from anterior mesoderm, and is required specifically for formation of the lateral aortae.

Mutant huntingtin impairs vesicle formation from recycling endosomes by interfering with Rab11 activity.

Huntingtin (Htt) localizes to endosomes, but its role in the endocytic pathway is not established. Recently, we found that Htt is important for the activation of Rab11, a GTPase involved in endosomal recycling. Here we studied fibroblasts of healthy individuals and patients with Huntington's disease (HD), which is a movement disorder caused by polyglutamine expansion in Htt.

Negative guidance factor-induced macropinocytosis in the growth cone plays a critical role in repulsive axon turning.

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities.

Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes.

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M).

Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes.

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane.

The WD40 and FYVE domain containing protein 2 defines a class of early endosomes necessary for endocytosis.

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in approximately 30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-beta receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans, which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome.

Plasma membrane domains specialized for clathrin-mediated endocytosis in primary cells.

Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells.