Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on β-catenin activity, revealing a function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.

ABSTRACT Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, maturation and transfer. In melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional (convective) motion leading to their peripheral location in the cell, and are not fully matured. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on β-catenin activity, revealing a novel function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.

BACKGROUND:NRD convertase, also termed Nardilysin, is a Zn(++) metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined.METHODS:To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa.RESULTS:Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme.CONCLUSIONS:The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.