Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.

Abstract. Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 13 chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (13-GFP) in class II-expressing Mel JuSo cells. This modification of the class II 13 chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ 13-GFP chimera was studied in living cells at 37°C. We visualize rapid movement of acidic class II/13-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway. MHC class II molecules present peptides to CD4 ÷ T cells. Most bound peptides are derived from antigens degraded in the endosomal pathway. To allow association with these peptides, class II molecules are targeted to endosomal compartments by the invariant chain (or Ii) 1 (7,8). Here, Ii is degraded and Ii-degradation products are exchanged with antigenic peptides, a process catalyzed by 41,47,48). The endosomal compartments where class II molecules are loaded with peptide may be considered "special" lysosomes with a multilamellar and/or multivesicular appearance and were originally termed MIIC for MHC class II--containing compartments (31). This "unique" morphology appears to be induced by the expression of class II molecules (4). Although earlier endosomal compartments have been noted as "specialized class II loading compartments" as well (1, 50), HLA-DM and class II molecules are generally located in compartments with lysosomal proteins like CD63, lamp-l, and cathepsin D (13,24,32,42).

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and 'pinching off' of the limiting membrane into the luminal space [1] [2] [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large 'empty' vacuoles.

Rho GTPases are crucial regulators of the actin cytoskeleton and they play a role in the control of membrane trafficking. In contrast to the close family members RhoA and RhoC, RhoB localises to endosomes and delays epidermal growth factor receptor traffic. Here, we show that activated RhoB induces the peripheral distribution of endosomes, which align along subcortical actin stress fibres and are surrounded by an actin coat. The Diaphanous-related formin, Dia1, is recruited to endosomes by activated RhoB. Dia1 is required for the formation of the actin coat around endosomes downstream of RhoB, connecting membrane trafficking with the regulation of actin dynamics.