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Reports on the 2003 meeting of the European Society for Pigment Cell Research in Ghent University in Gent, Belgium. Program of the meeting; List of speakers and topics discussed at the meeting.

To unravel the etiopathogenesis of café au lait spots in neurofibromatosis type 1 we study new possible protein-protein interactions of neurofibromin, gene product involved in NF1. Therefore, we performed yeast two hybrid screening (YTHS) against a commercially available human brain cDNA library (Clontech) and a human melanocyte (MC) cDNA library (MATCHMAKER Library Construction & Screening kit – Clontech). We amplified four relevant sequence segments of neurofibromin (GenBank n° NM_000267) and PCR products were inserted in a pBD-Gal4 vector (Stratagene) resulting in 4 bait plasmids: ‘‘bait 1’’ (part N-terminus: bp 51-391), ‘‘bait 2’’ (major part of the GAP-related domain (GRD): bp 4071-4671), ‘‘bait 3’’ (SEC-14 domain: bp 4495-4929) and ‘‘bait 4’’ (C-terminal part: bp 7629-8235). YTHS was performed using the yeast strain Saccharomyces Cerevisiae PJ69-4A, harboring HIS3, ADE and LacZ as reporter genes. Because of the known functional importance of the GRD, this was the first ‘‘bait’’ to be screened against both libraries. Screening of GRD bait 2 against the human brain cDNA library resulted in 3 possible relevant interactions: aldolase C (an enzyme important in glycolysis, known to bind F-and G-actin and intermediate filaments), cyclin I (a cell-cycle protein isolated from human brain) and amyloid precursor protein (APP) (a ubiquitously expressed transmembrane glycoprotein important in Alzheimer's disease and cutaneous wound repair). Screening against the human MC cDNA library resulted in 2 interactions: hypothetical protein HSPC016, with unknown function, and COP9 subunit 6 (Mov-34 homolog) described as being related to a subunit of the human eukaryotic initiation factor 3 and homologous to a 26S proteasome subunit. The interactions picked up are now being confirmed by colocalisation and coimmunoprecipitation assays in melanocytes. The neurofibromin protein is known to interact with microtubules and intermediate filaments and to form a complex with the microtubule motor protein kinesin-1. APP is required for kinesin-mediated axonal transport and binds directly to the kinesin light chain. According to our preliminary results APP also directly interacts with the neurofibromin GRD. This could reflect a new function of neurofibromin in vesicular transport. Relevance of the other interactions is under study now. [ABSTRACT FROM AUTHOR]

Rab GTPases, which are Ras-like small GTPases, are involved in microtubule and actin dependent intracellular vesicle movement and are key players in regulation of endo-and exocytosis. Rab27a and rab27b, members of the rab27 subfamily, show 72% amino acid sequence homology and are functionally redundant in platelets and endocrine cells. We found that rab27b is highly expressed in several human melanoma cell lines but very lowly in human primary melanocytes. Differential expression of rab27b in melanoma cell lines and its potential role in tumour progression remains poorly understood. Here we show that over-expression of enhanced green fluorescent protein (eGFP) tagged rab27b induced aggressive invasion of the human non invasive VKR melanoma cell line, invasive VQL melanoma cell line and primary melanocytes into collagen type I whereas eGFP tagged rab27a and eGFP did not. Invasion of eGFP-rab27b melanocytes was accompanied by stress fibber formation and alteration of the dendritic phenotype. Pull down assays demonstrated that active Rho was up regulated in eGFP-rab27b melanocytes. Co-expression of Rac1N17 RhoAN19 with eGFP rab27b counteracted rab27b-induced invasion, suggesting a requirement for Rac and Rho activity in the invasion process. Rab27b dependent invasion was also antagonized by introduction of a dominant negative myosin Va construct, which is known to interact with rab27b positive vesicles. We conclude that rab27b specific expression stimulates invasion and migration of melanoma cell lines and primary melanocytes into collagen type I by Rac/Rho signaling. [ABSTRACT FROM AUTHOR]

Patients with the autosomal recessive Griscelli-Pruniéras syndrome display a lethal immunologic and neurologic disorder, accompanied by very unusual silvery-gray colour of the scalp hair, eyelashes and eyebrows but only partial albinism of the skin. In a minority of the patients a mutation in the myosin Va gene, an actin-associated ATPase implicated in organelle movement, was discovered. The majority of the patients displayed a mutation in the rab27a gene, a small GTPase that is associated with the membrane of melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Here we discuss the genomic rab27a deletion found in a 21 months old Griscelli patient. We also provide evidence that the loss of functional rab27a in these Griscelli melanocytes is partially compensated by the up-regulation of rab27b, a small GTPase that is closely related to rab27a. By quantitative real time PCR we find that rab27b is lowly expressed in normal primary melanocytes but is significantly up-regulated in melanocytes derived from the Griscelli patient. By using immuno-fluorescence confocal microscopy and immuno-electron microscopy we were able to show that rab27b is associated with the melanosome membrane of melanocytes. Yeast two-hybrid screening revealed that rab27b, as described for rab27a, can form a tripartite complex with melanophilin, a rab27a effector, and protein products of myosin Va transcripts that do contain exon F. Our data suggests that the up-regulated rab27b in Griscelli melanocytes can partially take over the function of rab27a. This could explain the fact that our Griscelli patient displayed only partial albinism in the skin. [ABSTRACT FROM AUTHOR]