Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.

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Autophagy is a membrane trafficking mechanism that delivers cytoplasmic cargo to the vacuole/lysosome for degradation and recycling. In addition to non-specific bulk cytosol, selective cargoes, such as peroxisomes, are sorted for autophagic transport under specific physiological conditions. In a nutrient-rich growth environment, many of the autophagic components are recruited for executing a biosynthetic trafficking process, the cytoplasm to vacuole targeting (Cvt) pathway, that transports the resident hydrolases aminopeptidase I and α-mannosidase to the vacuole in Saccharomyces cerevisiae. Recent studies have identified pathway-specific components that are necessary to divert a protein kinase and a lipid kinase complex to regulate the conversion between the Cvt pathway and autophagy. Downstream of these proteins, the general machinery for transport vesicle formation involves two novel conjugation systems and a putative membrane protein complex. Completed vesicles are targeted to, and fuse with, the vacuole under the control of machinery shared with other vacuolar trafficking pathways. Inside the vacuole, a potential lipase and several proteases are responsible for the final steps of vesicle breakdown, precursor enzyme processing and substrate turnover. In this review, we discuss the most recent developments in yeast autophagy and point out the challenges we face in the future.

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Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles. The molecular mechanism of autophagy has been extensively studied using the yeast, Saccharomyces cerevisiae, during these past 10 years. These studies suggested that the molecular machinery of autophagosome formation is well conserved from yeast to higher eukaryotes. Identification and characterization of the mammalian counterparts of the yeast autophagy proteins has facilitated our understanding of mammalian autophagy, particularly of autophagosome formation. These findings are now being applied to studies on the physiological roles of autophagy in mammals.

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Macroautophagy is a major lysosomal catabolic process conserved from yeast to human. The formation of autophagic vacuoles is stimulated by a variety of intracellular and extracellular stress situations including amino acid starvation, aggregation of misfolded proteins, and accumulation of damaged organelles. Several signaling pathways control the formation of autophagic vacuoles. As some of them are engaged in the control of protein synthesis or cell survival this suggests that macroautophagy is intimately associated with the execution of cell proliferation and cell death programms. Whether or not these different signaling pathways converge to a unique point to trigger the formation of autophagic vacuole remains an open question.

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Lysosomes are ubiquitous organelles that carry out essential household functions. Certain cell types, however, contain lysosome-related organelles with specialized functions. Their specialized functions are usually reflected by specific morphological and compositional features. A number of diseases that develop due to genetic mutations, pathogen exposure or cell transformation are characterized by dysfunctional lysosomes and/or lysosome-related organelles. In this review we highlight adaptations and malfunction of the endosomal/lysosomal system in normal and pathological situations with special focus on MHC class II compartments in antigen presenting cells and melanosomes in pigment cells.

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Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinα were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importin α beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinα membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importin α membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-α or γ-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.

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Primary cultures of heart muscle cells provide powerful tools for cardiac cell biological research that permits both physiological and biochemical approaches. In the present study we analyzed the endocytosis of cardiac cells and presented morphological characterization of the endocytic machinery using markers, which enabled us to follow the fluid-phase, receptor-mediated endocytosis and the internalization of large particles. Our results demonstrated the route of the internalized cargo to early endosomes followed or not by its discharge in the late compartments. We also confirmed the ability of cardiac muscle cells to ingest large particles such as the mannosylated ligand zymosan A, and even internalize whole eukaryotic cells such as the protozoan parasite Trypanosoma cruzi. Since endocytosis is involved in many important cellular functions, the present work contributes to the knowledge of possible additional roles played by cardiac muscle cells besides their well known ability to act as physically energetic cells in the body, constantly contracting without tiring.

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Nonmuscle myosin II activity is regulated by phosphorylation of the myosin II regulatory light chain (MRLC) at Ser19 or at both Thr18 and Ser19, and the phosphorylation of MRLC promotes the contractility and stability of actomyosin. To analyze the states of MRLC phosphorylation at the leading edge in the motile HeLa cells, we have examined the subcellular distribution of monophosphorylated or diphosphorylated form of MRLC using a confocal microscope. The cross-sectional imaging revealed that monophosphorylated MRLC distributed throughout the cortical region and the leading edge, but its fluorescent signal was much stronger at the leading edge. This distribution pattern of monophosphorylated MRLC was almost identical to those of myosin II and F-actin. On the other hand, diphosphorylated MRLC is localized at the base of leading edge, spatially very close to the substrate, and colocalized with F-actin in part at the base of filopodia. Diphosphorylated MRLC was hardly detectable at the tip of filopodia and the cell cortical region, where monophosphorylated MRLC was clearly detected. These localization patterns suggest that myosin II is activated at the leading edge, especially at the base but not the tip of filopodia in motile cells. Next, we analyzed the cells expressing GFP-tagged recombinant MRLCs. Expression of GFP-tagged diphosphorylatable and monophosphorylatable MRLCs led to a significant increase in the filopodial number, compared with the cells expressing nonphosphorylatable MRLC. This result indicated that expression of phosphorylatable MRLC enhances the formation of filopodia at the wound edge.

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