Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration.

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of ß1-integrin by interference with recycling following normal endocytosis of inactive ß1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces ß1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG.

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.

Anisotropic nanocrystal arrays organized on protein lattices formed by recombinant clathrin fragments.

Recombinant clathrin protein fragments form assemblies that template gold nanocrystals in an array across the latticed surface. The nanocrystals exhibit unusual anisotropic morphologies with long range ordering, both of which are dependent upon the presence of a hexahistidine tag on the clathrin heavy chain fragments.

Intracellular processing of riboflavin in human breast cancer cells.

A variety of polarized epithelial cells, such as human breast cancer (MCF-7), have mechanistically evolved the ability to adapt to the dynamic cellular environment and maintain homeostasis of an array of micronutrients which display conditional requirements. Active absorption mechanisms, including endocytosis, are able to control cell surface recognition and protein expression which are associated with a substance's intracellular processing and kinetics. Riboflavin (RF), or vitamin B2, has been recognized as an important factor in a multitude of terminal disease states, most notably in breast cancer, where its cellular absorption is significantly enhanced. In order to delineate the regulatory mechanisms and kinetics associated with RF control in human breast cancer tissue, this study aimed to model its absorption profile and identify its intracellular regulatory components. Using both the Michaelis-Menten equation and a modified version of it, incorporating both active internalization and passive diffusion, RF absorption displayed better correlation ( r (2) > 0.998) with the mixed, active and passive, model exhibiting kinetic parameters characteristic of a receptor-mediated uptake mechanism ( J max = 2.58 pmol/5 min, K m = 106 nM) at extracellular RF concentrations under 5 muM and a passive component existing at RF concentrations greater than 5 muM. Following internalization, RF was able to recycle back to the membrane with a half-life of 13.7 min at 37 degrees C, which occurred more rapidly with increasing extracellular RF concentrations ( t 1/2 = 5.4 min at 1 muM) and decreasing temperatures ( t 1/2 = 6.4 min at 4 degrees C). Furthermore, modification to endosomal pH using the lysomotropic agents monensin (25 muM) and primaquine (300 muM) significantly inhibited the exocytosis of RF (61 and 30% of control), whereas biochemical modification of endocytic trafficking with okadaic acid (1 muM) led to a significant increase in RF exocytosis (208%). In conclusion, RF homeostasis in MCF-7 cells is a well regulated process which is dependent upon RF concentration, temperature, and endosomal acidification.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PURPOSE: To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. MATERIALS AND METHODS: PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. RESULTS: Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. CONCLUSION: These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

Dynamin 2 regulates riboflavin endocytosis in human placental trophoblasts.

Riboflavin is thoroughly established to be indispensable in a multitude of cellular oxidation-reduction reactions through its conversion to coenzyme forms flavin mononucleotide and flavin adenine dinucleotide. Despite its physiological importance, little is known about specific mechanisms or proteins involved in regulating its cellular entry in humans. Studies involving biochemical modulators and immunological inhibition assays have indirectly revealed that riboflavin internalization and trafficking occurs at least in part through a clathrin-dependent receptor-mediated endocytic process. Here, using a two-tiered strategy involving RNA interference and the overexpression of dominant-negative constructs, we directly show the involvement of this endocytic mechanism through the requirement of the pluripotent endocytic vesicle scission enzyme, dynamin 2 GTPase, in human placental trophoblasts. Similar to the endocytic control ligand, transferrin, riboflavin is shown to exhibit 50% dependence on the functional expression of dynamin 2 for its active cellular entry. Furthermore, this reduced vitamin uptake correlates with >2-fold higher riboflavin association at the cell surface. In addition, fluorescent ligand endocytosis assays showing colocalization between rhodamine-riboflavin and the immunostained caveolar coat protein, caveolin 1, suggest that the active absorption of this important nutrient involves multiple and distinct endocytosis pathways.

cAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process.

Riboflavin (RF, vitamin B(2)), an essential micronutrient central to cellular metabolism through formation of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors, is internalized, at least in part, via a proposed receptor-mediated endocytic (RME) process. The purpose of this study was to delineate the cellular RF distribution using human placental trophoblasts and evaluate the regulatory role of cAMP in this process. Subcellular fractionation and three-dimensional confocal microscopy analyses were carried out to define the RF accumulation profile. Biochemical assays evaluating the cAMP dependence of this pathway were also performed. This study records an intracellular RF distribution pattern that shows dynamic accumulation of the ligand predominantly in the endosomal and lysosomal compartments and to a lesser extent in the Golgi and mitochondria. In contrast, transferrin (TF) colocalizes rapidly within endosomes with minimal accumulation in the other organelles. The temporal and spatial distribution of RF and TF colocalized with unique markers of the endocytic machinery provides added morphological evidence in support of the RME process with ultimate translocation to the mitochondrial domain. Colocalized staining with the Golgi also suggests a possible recycling or exocytic mechanism for this ligand. Furthermore, this study demonstrates cAMP regulation of the putative ligand-bound RF receptor and its association into endocytic vesicles. Delineating the dynamics of the process governing cellular RF homeostasis presents an untapped resource that can be further exploited in improving our current understanding of nutritional biology and fetal growth and development, and perhaps in targeting the endogenous system for developing novel therapeutic approaches.

Efflux of depsipeptide FK228 (FR901228, NSC-630176) is mediated by P-glycoprotein and multidrug resistance-associated protein 1.

Depsipeptide FK228 [(E)-(1S,4S,10S,21R)-7[(Z)-ethylideno]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone], a novel histone deacetylase (HDAC) inhibitor, previously was reported to be a P-glycoprotein (Pgp) substrate. We now expand the investigation to demonstrate that FK228 is a substrate for Pgp and multidrug resistance-associated protein 1 (MRP1). Transport of FK228 across the Caco-2 cell monolayer in apical to basolateral (AP-->BL) and basolateral to apical (BL-->AP) directions in the absence and presence of Pgp and MRP inhibitors were investigated. An in vitro uptake study in human red blood cells (RBCs) and a cytotoxicity assay in MRP1(-) HL60 and MRP1(+) HL60Adr cells were conducted to show that FK228 is an MRP1 substrate. An FK228-resistant cell line (HCT15R) was developed from HCT15 colon carcinoma and characterized using a 70-oligomer cDNA microarray, reverse transcription-polymerase chain reaction, Western blot analysis, histone acetyltransferase (HAT) and HDAC activity assays, and cytotoxicity assays. FK228 showed a nearly unidirectional flux across the Caco-2 cell monolayer, with the BL-->AP apparent permeability coefficient (P(app)) 32 times that of AP-->BL without apparent saturation. Pgp inhibition decreased the BL-->AP P(app) and increased the AP-->BL P(app). RBC showed a concentration-dependent uptake and saturable efflux of FK228. HL60Adr cells were 4-fold more resistant to FK228 than HL60 cells, and the resistance was reversed by MRP inhibition. Up-regulation of Pgp, but not changes of MRPs or HAT/HDAC enzymatic activities, was the major mechanism for the acquired FK228 resistance. These studies demonstrate that FK228 is a substrate for Pgp and MRP1, and reversible Pgp up-regulation is predominantly involved in FK228 resistance in vitro.

A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.

Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.

Current perspectives on the cellular uptake and trafficking of riboflavin.

The role of riboflavin in cell maintenance and growth, and the mechanism by which it is absorbed into various human tissues and cell lines has been extensively studied over the past decade. Evidence suggests two absorption mechanisms, a saturable-active component that dominates at near physiological vitamin concentrations and a passive component that is revealed at oversupplemented riboflavin conditions. Various transport modulator studies consistently suggest a highly riboflavin specific, temperature-dependent active transport mechanism that is regulated by the Ca2+/calmodulin pathway. The PKA and PKG pathways have also been implicated in absorption regulation. The long-standing model that riboflavin absorption involves a carrier-mediated transporter has recently been challenged through studies suggesting a receptor-mediated endocytic component. The presence of a soluble, human riboflavin binding protein in the transport stratagem has been shown to play an important role in fetal development. The relationship of this binding protein with the riboflavin specific membrane bound protein, though currently not well defined, may involve a protein-protein interaction that plays a primary role in this proposed receptor-mediated component.

Cytoskeletal motors and cargo in membrane trafficking: opportunities for high specificity in drug intervention.

Membrane trafficking comprises the directed transport of vesicle and/or organelle cargos to specific locations throughout the cell, and is primarily driven by molecular motors tracking along microtubules and microfilaments. The mechanisms by which specific motor complexes attach to their respective vesicular cargo is of great interest, and is only now starting to be unraveled. The proteins identified as links between the molecular motors and the vesicular cargo are viable drug targets and represent opportunities to regulate small groups of related proteins or even single proteins, such as receptors and transporters, at the cytosolic trafficking level. Ultimately, continued development in this area will lead to greater success in directing endocytosed drugs to the desired intracellular targets, such as the cell nucleus or the basolateral membrane.

Microfabricated porous silicon particles enhance paracellular delivery of insulin across intestinal Caco-2 cell monolayers.

PURPOSE: Novel porous silicon microparticles were fabricated and loaded with fluorescein isothiocyanate (FITC)-insulin, a model hydrophilic pharmacologically active protein, along with varied doses of sodium laurate (C12), a well-known permeation enhancer. METHODS: Particle and liquid formulations were compared as a function of apical to basolateral flux of FITC-insulin across differentiated human intestinal Caco-2 cell monolayers grown on Transwell inserts. RESULTS: The flux of FITC-insulin from silicon particles across cell monolayers was nearly 10-fold higher compared with liquid formulations with permeation enhancer and approximately 50-fold compared with liquid formulations without enhancer. By increasing C12 dose per particle with a concomitant decrease in total particles added per monolayer, the percent of FITC-insulin transport resulted in a linear increase up to 25% monolayer coverage. CONCLUSIONS: Although maintaining monolayer integrity and transepithelial electrical resistance, maximum drug transport (20%/h) was achieved with 0.337 microg C12 dose per particle, and total particle loading at 25% monolayer coverage.