Automatic detection and analysis of cell motility in phase-contrast time-lapse images using a combination of maximally stable extremal regions and Kalman filter approaches.

Phase-contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase-contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase-contrast images in time-lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time-lapse movies, the MSER-based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase-contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time-consuming large-scale dynamical analysis of cultured cells.

A hexavalent Coxsackievirus B vaccine is highly immunogenic and has a strong protective capacity in mice and nonhuman primates.

Coxsackievirus B (CVB) enteroviruses are common human pathogens known to cause severe diseases including myocarditis, chronic dilated cardiomyopathy, and aseptic meningitis. CVBs are also hypothesized to be a causal factor in type 1 diabetes. Vaccines against CVBs are not currently available, and here we describe the generation and preclinical testing of a novel hexavalent vaccine targeting the six known CVB serotypes. We show that the vaccine has an excellent safety profile in murine models and nonhuman primates and that it induces strong neutralizing antibody responses to the six serotypes in both species without an adjuvant. We also demonstrate that the vaccine provides immunity against acute CVB infections in mice, including CVB infections known to cause virus-induced myocarditis. In addition, it blocks CVB-induced diabetes in a genetically permissive mouse model. Our preclinical proof-of-concept studies demonstrate the successful generation of a promising hexavalent CVB vaccine with high immunogenicity capable of preventing CVB-induced diseases.

Isoproterenol-induced redistribution of endosomes in cardiac myocytes.

During myocardial infarction, it is well known that the cytoplasm of cardiac myocytes becomes more acidic due to lactate accumulation. The resulting necrosis is believed to result, at least in part, from the leakage of lysosomal enzymes into the cytoplasm. In addition, it has previously been shown that cytoplasm acidification in tissue culture cells can cause the redistribution of late endosomes and tubular lysosomes. In the present study, we have investigated whether lysosomal/endosomal structures were affected during isoproterenol-induced infarct-like myocardial necrosis in rat heart in vivo. In parallel, we treated rat primary myocyte cultures with a high dose of isoproterenol or low pH. We followed the fate of lysosomal enzymes, a lysosomal membrane glycoprotein and the cation-independent mannose 6-phosphate receptor. Lysosomes were intact until irreversible injury became evident suggesting that the lysosomal enzyme release concomitant with the leakage of cytoplasmic enzymes is merely a consequence of cell death. During the early phase of injury, when the myocyte cytoplasm was mildly acidified, late endosomes showed fragmentation and microtubule-dependent movement towards the periphery, while the subcellular distribution of lysosomes was unchanged. Both processes were also observed after mild artificial acidification of the cytoplasm. Our data show that late endosomes and lysosomal trafficking are affected early during isoproterenol-induced myocardial injury causing a pH-dependent redistribution of late endosomes.

3-Methyladenine inhibits transport from late endosomes to lysosomes in cultured rat and mouse fibroblasts.

The effect of 3-methyladenine on transport from endosomes to lysosomes was studied in rat embryonic and mouse 3T3 fibroblasts. Subcellular fractionation in 27% Percoll gradients showed that the pre-endocytosed (5 min pulse) horseradish peroxidase (HRP) was not transported from endosomes to dense lysosomes in cells chased in the presence of 10 mM 3-methyladenine. However, fractionation in 20% Percoll gradients, which separated early endosomes from late endosomes and lysosomes, as well as light and electron microscopic experiments, showed that HRP was transported from early endosomes to the perinuclear late endosomes. Immunoprecipitation of metabolically labeled cells was used to study the biosynthetic processing of a lysosomal proteinase, cathepsin L. The results showed that the early processing of the precursor to the intermediate form was not affected by 3-methyladenine, while the late processing of the intermediate to the mature form was retarded. In addition, immunofluorescence labeling showed that 3-methyladenine treatment caused accumulation of cathepsin L in the perinuclear area. Another lysosomal enzyme, beta-glucuronidase, was normally distributed in both perinuclear and peripheral vesicles which indicated that the localization of lysosomes was not altered. The results thus suggest that the late processing of cathepsin L was inhibited because transport from perinuclear endosomes to lysosomes was retarded. In conclusion, both endocytic pulse-chase experiments and immunoprecipitation of metabolically labeled cathepsin L indicate that 3-methyladenine inhibits transport from late endosomes to mature lysosomes in both rat and mouse fibroblasts.

At reduced temperature, endocytic membrane traffic is blocked in multivesicular carrier endosomes in rat cardiac myocytes.

Temperatures around 20 degrees C are known to block degradation of endocytosed material by preventing its transport to lysosomes, accordingly reduced temperature has been widely used to define endosomes. Newer studies have revealed that the low temperature block is proximal to perinuclear late endosomes, but it is not clear whether the block is already in early endosomes, or whether the traffic proceeds to multivesicular carrier endosomes which mediate transport from early to late compartments. We have now focused on this problem using rat cardiac myocytes. First, cell fractionation on Percoll gradients showed that at reduced temperatures (22 degrees C and 26 degrees C), with prolonged chase periods, endocytosed horseradish peroxidase was able to proceed from early endosomes to later compartments but not up to lysosomes. Further, microscopic experiments with fluorescent endocytic marker FITC-dextran showed that the marker did not accumulate in the perinuclear area, as was the case at 37 degrees C, but stayed in peripheral cytoplasm at reduced temperatures, even after 16-h chase. Second, electron microscopic pulse labeling showed that, at 22 degrees C, endocytosed gold particles (BSA-gold) are transported to compartments not accessible to HRP internalised later to early endosomes. Thus, these gold particles had reached a later compartment. Morphologically these vesicles were multivesicular bodies of 0.5-1 microm in diameter. Third, we used fluorescence microscopy to study the effect of reduced temperature on transferrin uptake and recycling. At 17 degrees C and 22 degrees C, transferrin was internalized normally to peripheral (sorting) and perinuclear (recycling) vesicles. If transferrin was first taken up at 37 degrees C, and the cells were then chased at various temperatures from 37 degrees C to 17 degrees C, the recycling was slowed down but not entirely blocked at the reduced temperatures. From these results we can conclude that (1) endocytic traffic is blocked in multivesicular carrier endosomes at and below 26 degrees C, and that (2) reduced temperature slows down transport in the recycling pathway, without a complete block.

Isoproterenol inhibits fluid-phase endocytosis from early to late endosomes.

We have shown recently that isoproterenol affects both the cellular location and the morphology of late endosomes in a pH-dependent manner [Marjomäki et al., Eur. J. Cell Biol. 65, 1-13 (1994)]. In this study, using fluorescence and quantitative electron microscopy, we wanted to examine further what is the fate of internalized markers during their translocation from early to late endosomes under isoproterenol treatment. Fluorescein dextran internalized for 30 min (10-min pulse followed by a 20-min chase) showed accumulation in the cellular periphery during isoproterenol treatment in contrast to the control cells, which accumulated dextran in the perinuclear region. Quantitative electron microscopy showed that the markers accumulated in the early endosomes and putative carrier vesicles. In addition, different particulate markers that were internalized sequentially accumulated in similar structures due to the isoproterenol treatment, altogether suggesting that isoproterenol retards the translocation of markers to the later structures. Prelabelling of the late endosomes with fluorescent dextran or BSA-coated gold particles showed that isoproterenol causes a reduction of the mean size of the prelabelled late endosomes as well as a shift of these vesicles to the cellular periphery. Isoproterenol had no apparent effect on the morphology nor on the location of lysosomes. Percoll fractionation showed that the changes in late endosomal location and morphology did not change their characteristic density. Furthermore, electron microscopy showed that, in the cellular periphery, these late endosomal elements did not fuse with early endosomal structures, which is in agreement with the results of biochemical in vitro cell-free assays carried out by others. In conclusion, the results show that isoproterenol inhibits transport from early to late endosomes in a manner that may be pH- and/or Ca(2+)-dependent. Simultaneously, isoproterenol causes fragmentation of the late endosomal compartment and the shift of these fragments to the cellular periphery, where they have a restricted ability to fuse with earlier endosomal structures.

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes.

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.

Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysosomal trafficking in rat cardiac myocytes.

By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. MPR was associated mostly with inner membranes, whereas lgp120 was predominantly bound to the outer limiting membrane. MPR, lgp120, and MEP were not detected in Golgi stacks, but some labeling was seen in the putative TGN. Our data suggest that the MPR structures are prelysosomes involved in lysosomal enzyme targeting in rat cardiac myocytes.

Recombinant avidin and avidin-fusion proteins.

Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.

Morphological and enzymatic heterogeneity of suramin-induced lysosomal storage disease in some tissues of mice and rats.

Suramin-induced lysosomal storage disease reproduced in the rat was extended to the mouse with the attempt to characterize enzymatically and morphologically heterogeneous responses of various organs to the drug. Suramin administration strikingly decreased (3-6 days afterward) the activity of beta-glucuronidase in all tissues studied (kidney, liver, heart, and skeletal muscle). The enzymatic responses were small in the activities of beta-N-acetyl-glucosaminidase. The activity of arylsulfatase A decreased to a varying degree in mouse tissues, but in rats the activity increased in liver and skeletal muscle. The activity of cathepsin D increased in rat tissues. Suramin induced morphological changes characteristic to lysosomal storage diseases in kidney and liver but not in heart and skeletal muscle of both mice and rats. Kidney was appreciably more susceptible to suramin than liver. The occurrence of lysosomal accumulations, membranous lamellar inclusions, and granular material were most prominent in tubular cells of kidney and in Kupffer cells of liver. These cells also presented intensive Alcian blue staining. Interestingly, the enzymatic and morphological responses did not correlate with each other, which may reflect differences in the regulation of lysosomal functions in various cell types.

Phosphomannosyl receptors of lysosomal enzymes in cardiac and skeletal muscles of young and old mice.

The endogenous activity and the binding of high-uptake beta-N-acetylglucosaminidase were assayed in the membranes of heart and skeletal muscles of young (2 months) and old (15 months) NMRI-mice (Mus musculus) to evaluate the age-related changes in the phosphomannosyl receptors of lysosomal enzymes in muscular membranes. The total activities of beta-N-acetylglucosaminidase were significantly higher in cardiac and skeletal muscles of old than young mice. The total and the specific (inhibited by mannose-6-phosphate) binding of beta-N-acetylglucosaminidase to the membranes of cardiac muscle, but not to those of skeletal muscle, were higher in old mice than in young ones. The endogenous activity of beta-N-acetylglucosaminidase was significantly higher in the membranes of skeletal muscles of old mice than in those of young mice. The membranes of heart muscles did not show any difference in the endogenous activities. The saturation properties of the binding of beta-N-acetylglucosaminidase to the phosphomannosyl receptors were very similar in the membranes of heart and skeletal muscles of both age groups. We conclude that during aging the number of phosphomannosyl receptors of lysosomal enzymes increases in the membranes of heart muscle while the occupancy of phosphomannosyl receptors with endogenous ligands increases in the membranes of skeletal muscle.

Effects of free fatty acids, lysophosphatides and phospholipase treatment on lipid peroxidation of myocardial homogenates and membrane fractions.

The effects of various free fatty acids, lysophosphatides and phospholipase treatments on the enzymatic and the non-enzymatic lipid peroxidation capacities in the heart homogenates and subcellular fractions were studied. The results showed a dose related inhibition of both the enzymatic and non-enzymatic lipid peroxidation with free fatty acids. A significant inhibition occurred as early as at the concentration of 25-50 microM of several fatty acids both in homogenates and in organelle fractions. In general, the inhibition was greatest with cis-unsaturated, long-chain fatty acids. The inhibition was also induced by the pretreatment of the homogenates with phospholipase A2 but not with phospholipase C. The lysophosphatidyl cholines (16:0 and 18:1) had a stimulatory effect on the enzymatic lipid peroxidation capacity at the physiological concentrations. The results show that the stimulatory/inhibitory effect of various lipid amphiphiles on lipid peroxidation is strongly structure linked and the mitochondrial fraction is the most susceptible to the injury induced by lipid amphiphiles.

Characteristics of lysosomal phosphomannosyl-enzyme receptors in the rat heart.

The receptor system recognizing mannose 6-phosphate groups of lysosomal enzymes has been characterized, e.g. in fibroblasts and liver cells. The purpose of this study was to demonstrate the presence of a phosphomannosyl receptor system in rat heart muscle. The characterization of receptors was accomplished with beta-N-acetylglucosaminidase (beta-GA) secreted by rat embryo fibroblasts after ammonium chloride stimulation. The receptor binding of ligand enzymes was saturated by adding increasing concentrations of beta-GA and the binding increased linearly when the content of membrane protein was increased. The binding of beta-GA was inhibited by mannose and glucose phosphates, especially mannose 6-phosphate. Mannose itself did not inhibit binding of the enzyme, showing that the binding was not mediated by mannose receptors. Alkaline phosphatase treatment of beta-GA decreased the binding of ligand enzymes to receptors. Alkaline conditions increased the dissociation of receptor-ligand complexes, whereas the dissociation was minimal between pH 5.5 and 6.5. The proportion of endogenous beta-GA activity in membranes, probably representing receptor-bound location, varied between 40 and 55% of the total activity in various parts of rat cardiac muscle. The differences in the content of phosphomannosyl receptors, however, were insignificant between various cardiac muscle samples. At the organelle level the highest specific binding capacity, as well as the highest endogenous beta-GA activity, was in the sarcolemmal fraction. These results suggest that phosphomannosyl receptors also function in the endocytosis and transport of lysosomal enzymes in cardiomyocytes, as well as in several other cell types studied.

Phosphomannosyl receptors of lysosomal enzymes of skeletal muscle in neuromuscular diseases.

The phosphomannosyl receptor system is responsible for both the receptor-mediated endocytosis and the intracellular transport of lysosomal enzymes. In the present study this receptor system was examined in affected muscles of patients with various neuromuscular diseases. The total activity of beta-N-acetyl-glucosaminidase, a marker enzyme of lysosomal hydrolases, was significantly elevated in the patients with myopathies (polymyositis and muscular dystrophies) but only slightly increased in those with neurogenic muscle atrophies (amyotrophic lateral sclerosis, polyneuropathy or other neurogenic muscle disease). The increase was most prominent in the group of polymyositis. The content of phosphomannosyl receptors was increased in the patients with myogenic muscle disease but not in those with neurogenic disease. The receptor binding of lysosomal enzymes was saturable and inhibited with mannose 6-phosphate showing the typical characteristics of phosphomannosyl receptors. The characteristics of the receptors were very similar both to control and to diseased muscle samples. When surveying all the material, the content of phosphomannosyl receptors correlated highly significantly with the muscular activity of beta-N-acetylglucosaminidase, muscle atrophy index, and serum creatine kinase activity.

Entry of human parechovirus 1.

Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes alpha(V) integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against alpha(V) and beta(3) integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and cis-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.

Intracellular route of canine parvovirus entry.

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18 degrees C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.

The transmembrane protein p23 contributes to the organization of the Golgi apparatus.

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

Endocytosis in skeletal muscle fibers.

Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, labeled with transferrin, appeared as perinuclear and interfibrillar dots that partially colocalized with the GLUT4 compartment. Low-density lipoprotein, a marker of the lysosome-directed pathway, was transported into sparsely distributed perinuclear and interfibrillar dots that contacted microtubules. A majority of these dots did not colocalize with internalized transferrin, indicating that the recycling and the lysosome-directed pathways were distinct. In conclusion, the I band areas were active in endocytosis along the whole length of the multinucleated myofibers. The sorting endosomes distributed in a cross-striated fashion while the recycling and late endosomal compartments showed perinuclear and interfibrillar localizations and followed the course of microtubules.