Biology subjects

Ramalho, J. S.

Publications and source records attributed to Ramalho, J. S..

3 recordsLinked to original sources

bioRxiv2021

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin released by virtually all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs and proteins between different cells, tissues or organs. However, the mechanisms that regulate the selective loading of cytosolic proteins into these vesicles are still largely unknow. Here we describe a mechanism whereby proteins containing a pentapeptide sequence, biochemically related to the KFERQ-motif, are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, this mechanism is independent of the ESCRT machinery components TSG101 and VPS4b and dependent on HSC70, CD63, Alix, Syntenin-1, Rab31 and ceramides. The transcription factor and master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. Additionally, by tagging fluorescent proteins with KFERQ-like sequences we were able to follow inter-organ transfer of exosomes in zebrafish larvae. Our findings identify LAMP2A as a key component in exosome biogenesis while opening new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

cell biology↗

bioRxiv2021

Rab3a regulates melanin exocytosis induced by keratinocyte-conditioned medium

Skin pigmentation relies on melanin and is crucial for photoprotection against ultraviolet radiation-induced toxicity. Melanin is synthesized and stored in melanosomes, within melanocytes and then transferred to keratinocytes. While the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain poorly characterized. Previously, our group proposed that coupled exo/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for Rab11b and the exocyst tethering complex in this process. Using a fluorescence-based assay, we show here that keratinocyte-conditioned medium (KCM) specifically induces melanin exocytosis from melanocytes. Moreover, we found that Rab3a, but not Rab11b, regulates melanin exocytosis upon KCM stimulation. In fact, melanosomes accumulate in melanocyte dendrites upon KCM stimulation, co-localizing with Rab3a mainly in the vicinity of the plasma membrane. Additionally, Rab3a silencing does not affect melanin transfer in melanocyte/keratinocyte co-cultures, in contrast with Rab11b depletion, indicating that Rab11b regulates non-KCM-stimulated melanin exocytosis. Thus, our results suggest the existence of at least two distinct routes of melanin exocytosis: one controlled by Rab11b and another Rab3a-dependent, stimulated by KCM. Furthermore, these results provide evidence that soluble factors secreted by keratinocytes can control skin pigmentation via induction of melanocyte signaling pathways that promote peripheral transport of melanosomes and a Rab3a-mediated exocytosis mechanism.

molecular biology↗

bioRxiv2021

Melanocore uptake by keratinocytes occurs through phagocytosis and involves Protease-activated receptor-2 activation

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred - either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1 and CtBP1/BARS, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Furthermore, we confirmed that melanocore, but not melanosome uptake is dependent on the Protease-activated receptor-2 (PAR-2) and found that PAR-2 can be specifically activated by melanocores. As skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

cell biology↗