Introduction Internalised molecules are sorted from resident proteins and lipids along the endocytic pathway, through a complex series of regulated processes. Besides the well-characterized signal motifs, micro-domains on membranes play an important role for efficient sorting of proteins and lipids (Hopkins et al., 1994; Raiborg et al., 2003; Stoorvogel et al., 1987). The transferrin receptor accumulates in tubules before being recycled to the plasma membrane, while the EGF receptor is sequestered in membrane invaginations that give rise to internal vesicles of multi-vesicular endosomes (MVEs, also named multi-vesicular bodies) before its degradation in lysosomes (Hopkins et al., 1994; Raiborg et al., 2003). Compelling evidence from several laboratories indicate that microtubules and associated molecular motors of the dynein and kinesin families are involved in the tubulation of organelles and thereby might contribute to sorting events (Koster et al., 2003; Roux et al., 2002). The participation of actin networks and myosins in different steps of the secretory and endocytic pathways were also investigated but the role of these networks and their associated motors as mechanical devices for sorting processes has only been studied recently. The acto-myosin generated forces could help to cluster the cargos prior to their transport. For example, the class VI myosin, which binds adaptors proteins containing PDZ domains, such as Dab2 or GIPC, has been proposed to cluster receptors with PDZ-binding motifs in clathrin-coated regions (Hasson, 2003). Acto-myosin tension might also drive morphological changes that could contribute to the formation of micro-domains involved in the sorting process. In this context, in Dictyostelium discoidum the knockout of two genes encoding class I myosins, myoA and myoB, leads to a disordered morphology of sorting recycling endosomes (Neuhaus and Soldati, 2000). We have previously reported that another myosin from class I, myosin Ib (Myo1b, also named myosin I alpha or Myr1), participates in the cytoplasmic distribution of endosomes and lysosomes and in the delivery of internalised molecules to lysosomes in mammalian cells (Cordonnier et al., 2001; Raposo et al., 1999). Despite the involvement of this myosin in the traffic along the endocytic pathway, Myo1b is unable to move endocytic compartments on actin filaments in vivo (Cordonnier et al., 2001). However, Myo1b, together with actin, might drive morphological changes of the endocytic compartments, similarly to myoA and myoB in Dictyostelium discoidum. It might also act with actin as a dynamic scaffold and be involved in the sorting of specific cargos. To investigate further the mechanism by which Myo1b contributes to the traffic of internalized molecules towards the lysosomes, we used the pigmented human melanoma cell line MNT-1, which displays a well defined endocytic system. In these cells, endosomes that were traced with gold-labelled bovine serum albumin 15 minutes post uptake and displayed bilayered clathrin coats and internal vesicles, are intermediates for proteins transported to lysosomes and for proteins involved in the biogenesis of melanosomes (Raposo 4823 Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes co- immunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes. Keys words: Actin cytoskeleton, Biogenesis of melanosomes, Membrane traffic, Endocytosis Summary Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes Laura Salas-Cortes 1, *, Fei Ye 2, *, Danièle Tenza 1 , Claire Wilhelm 3 , Alexander Theos 4 , Daniel Louvard 1 , Graça Raposo 1 and Evelyne Coudrier 1,‡ 1 Institut Curie, CNRS UMR144, 26 rue d’Ulm, 75248, Paris, Cedex 05, France 2 E363 INSERM, Faculté de Médecine, Necker, 156 Rue de Vaugirard, 75015, Paris, France 3 Laboratoire des milieux désordonnés et hétérogènes, Universite Pierre et Marie Curie, Boite 86, 4 Place Jussieu, 75252 Paris Cedex 05, France 4 Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3451 Walnut Street, Philadelphia, PA 19104, USA *These authors contributed equally to this work ‡ Author for correspondence (e-mail: coudrier@curie.fr) Accepted 2 August 2005 Journal of Cell Science 118, 4823-4832 Published by The Company of Biologists 2005 doi:10.1242/jcs.02607 Research Article Journal฀of฀Cell฀Science