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Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable oculocutaneous albinism, immunodeficiency, mild bleeding diathesis, and an accelerated lymphoproliferative state. Abnormal lysosome-related organelle membrane function leads to the accumulation of large intracellular vesicles in several cell types, including granulocytes, melanocytes, and platelets. This report describes a severe case of CHS resulting from paternal heterodisomy of chromosome 1, causing homozygosity for the most distal nonsense mutation (p.E3668X, exon 50) reported to date in the LYST/CHS1 gene. The mutation is located in the WD40 region of the CHS1 protein. The patient's fibroblasts expressed no detectable CHS1. Besides manifesting the classical CHS findings, the patient exhibited hypotonia and global developmental delays, raising concerns about other effects of heterodisomy. An interstitial 747 kb duplication on 6q14.2-6q14.3 was identified in the propositus and paternal samples by comparative genomic hybridization. SNP genotyping revealed no additional whole chromosome or segmental isodisomic regions or other dosage variations near the crossover breakpoints on chromosome 1. Unmasking of a separate autosomal recessive cause of developmental delay, or an additive effect of the paternal heterodisomy, could underlie the severity of the phenotype in this patient., (Published 2010 Wiley-Liss, Inc.)

Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment. Considering the contribution of the stromal environment, we developed a membrane-free single-cell and spheroid based complementary model to study cancer invasion through native collagen type-I matrices. Cell morphology is preserved during the assays allowing real time monitoring of invasion-induced changes in cell structure and F-actin organization. Combining these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type-I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 8 days) respectively.

HPS is an autosomal recessive disorder characterized by oculocutaneous albinism and prolonged bleeding. Eight human genes are described resulting in the HPS subtypes 1-8. Certain HPS proteins combine to form Biogenesis of Lysosome-related Organelles Complexes (BLOCs), thought to function in the formation of intracellular vesicles such as melanosomes, platelet dense bodies, and lytic granules. Specifically, BLOC-2 contains the HPS3, HPS5 and HPS6 proteins. We used phylogenetic footprinting to identify conserved regions in the upstream sequences of HPS3, HPS5 and HPS6. These conserved regions were verified to have in vitro transcription activation activity using luciferase reporter assays. Transcription factor binding site analyses of the regions identified 52 putative sites shared by all three genes. When analysis was limited to the conserved footprints, seven binding sites were found shared among all three genes: Pax-5, AIRE, CACD, ZF5, Zic1, E2F and Churchill. The HPS3 conserved upstream region was sequenced in four patients with decreased fibroblast HPS3 RNA levels and only one HPS3 mutation in the coding exons and surrounding exon/intron boundaries; no mutation was found. These findings illustrate the power of phylogenetic footprinting for identifying potential regulatory regions in non-coding sequences and define the first putative promoter elements for any HPS genes.

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking steps within cells. In melanocytes, the GTP-bound form of Rab27A associates with the membranes of mature fully-pigmented melanosomes through its geranylgeranyl group. Once attached, Rab27A recruits the downstream effector Melanophilin (Mlph) and the actin-dependent motor protein Myosin Va (MyoVa). The molecular Rab27A/Mlph/MyoVA tripartite complex, which links melanosomes to the peripheral actin network, is required to achieve melanosome transfer to surrounding keratinocytes in the epidermis. Here we report a novel homozygous missense mutation c.127G>A, p.G43S in exon 2 of the RAB27A gene of an Afghani GSII patient. Laser scanning confocal microscopy showed that the G43S mutation, which is located in the highly conserved switch I region of Rab27A, induces perinuclear localization of melanosomes in normal melanocytes, and fails to restore melanosomes to the actin-rich periphery in GSII melanocytes. Co-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins.

Lysosome-related organelles (LROs) are a heterogeneous group of vesicles that share various features with lysosomes, but are distinct in function, morphology, and composition. The biogenesis of LROs employs a common machinery, and genetic defects in this machinery can affect all LROs or only an individual LRO, resulting in a variety of clinical features. In this review, we discuss the main components of LRO biogenesis. We also summarize the function, composition, and resident cell types of the major LROs. Finally, we describe the clinical characteristics of the major human LRO disorders.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis resulting in melanosome dysfunction and absent platelet dense bodies. HPS patients have oculocutaneous albinism, bruising, and bleeding. HPS-5 results from deficiency of the HPS5 protein, a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). HPS5 has an unknown function and lacks homology to known proteins. We performed ultrastructural studies of HPS-5 melanocytes revealing predominantly early-stage melanosomes with many small 3,4(OH)2-phenylalanine-positive vesicles throughout the cell body and dendrites. These findings resemble the distinct ultrastructural features of HPS-3 melanocytes; HPS3 is also a BLOC-2 component. Immunofluorescence and immunoEM studies showed decreased TYRP1 labeling in the dendrites of HPS-5 melanocytes, and the overall abundance of TYRP1 was reduced. No substantial differences were observed in the distribution or abundance of Pmel17 in HPS-5 melanocytes. In normal melanocytes, endogenous tyrosinase colocalized with Pmel17 and TYRP1 in the perinuclear area and dendritic tips; this was much reduced in HPS-5 melanocytes, particularly in the tips. We conclude that early stage melanosome formation and Pmel17 trafficking are preserved in HPS5-deficient cells. Tyrosinase and TYRP1 are mistrafficked, however, and fail to be efficiently delivered to melanosomes of HPS-5 melanocytes.

An extended, highly consanguineous Israeli Bedouin family with at least 20 individuals exhibiting a unique phenotype of oculocutaneous albinism (OCA) was identified. All known OCA genes were excluded in this family. Electron microscopic analysis of platelets revealed absence of dense bodies, suggesting a diagnosis of Hermansky-Pudlak syndrome (HPS). HPS is a rare autosomal recessive disorder of lysosome-related organelle biogenesis, clinically characterized by OCA and platelet dysfunction, sometimes accompanied by other systemic pathologies. All human HPS genes (HPS1-8) and five genes corresponding to murine HPS models were evaluated. Haplotype analysis and homozygosity mapping of the HPS loci revealed linkage to chromosome 10 in the studied family. Subsequently, a novel insertion mutation, c.1066-1067insG was identified in HPS6. Most frameshift mutations generating premature termination codon cause mRNA nonsense mediated decay (NMD), while intronless genes like HPS6 are usually not monitored by NMD. Expression analysis revealed no mRNA decay in patient's fibroblasts, hence truncated protein is most probably produced. Confocal microscopy revealed abnormal distribution of LAMP-3 (lysosomal associated membrane protein-3) in fibroblasts from the patients, indicating abnormal trafficking of lysosomal lineage organelles. So far, a single HPS-6 patient phenotypically similar to HPS-3 and HPS-5 has been identified. The HPS-6 phenotype in the studied family is unique since it resembles OCA and not HPS. Therefore, our finding broadens the phenotypic definition of HPS. Two major genetic isolates of HPS-1 and HPS-3 patients were previously diagnosed in Puerto Rico. The extended Bedouin family is the largest isolate of non-Puerto Rican HPS patients.

Melanosomes are lysosome-related organelles that synthesize, store and transport melanin. In epidermal melanocytes, melanosomes mature and are transferred to surrounding keratinocytes, which is essential for skin and coat colour. Mouse coat colour mutants reveal a critical role for the small GTPase Rab27a, which recruits myosin Va through its effector protein melanophilin/Slac2a. Here we have studied how two different Rab GTPases control two motor proteins during subsequent phases in transport of melanosomes. We show that the small GTPase Rab7 mainly associates with early and intermediate stage melanosomes and Rab27a to intermediate and mature melanosomes. Rab27a is found in an active state on mature melanosomes in the tips of the dendrites. The Rab7-Rab7-interacting lysosomal protein-dynein pathway only controls early and intermediate stage melanosomes because the mature melanosomes lack Rab7 and associate with the actin network through Rab27a recruited MyoVa. Thus two Rab proteins regulate two different motor proteins, thereby controlling complementary phases in melanosome biogenesis: Rab7 controls microtubule-mediated transport of early and Rab27a the subsequent actin-dependent transport of mature melanosomes.

The neurofibromatosis type 1 (NF1) gene product, neurofibromin, is known to interact with Ras, thereby negatively regulating its growth-promoting function. Although this is a well-established interaction, the discovery of other neurofibromin interacting partners could reveal new functional properties of this large protein. Using yeast two-hybrid analysis against a brain cDNA library, we identified a novel interaction between the amyloid precursor protein and the GTPase activating protein-related domain of neurofibromin. This interaction was further analyzed in human melanocytes and confirmed by immunoprecipitation and colocalization studies. In addition, we observed a colocalization of amyloid precursor protein and neurofibromin with melanosomes. Amyloid precursor protein has been proposed to function as a vesicle cargo receptor for the motor protein kinesin-1 in neurons. This colocalization of amyloid precursor protein and neurofibromin with melanosomes was lost in melanocytes obtained from normal skin of a NF1 patient. We suggest that a complex between amyloid precursor protein, neurofibromin, and melanosomes might be important in melanosome transport, which could shed a new light on the etiopathogenesis of pigment-cell-related manifestations in NF1.

Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.

Background: Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172-176., Results: Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20 degrees C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution., Conclusion: HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.

Patients with the autosomal recessive Griscelli-Pruniéras syndrome type II are immunologically impaired and have an unusual silvery-grey hypopigmented colour of scalp hair, eyelashes and eyebrows but no noteworthy pigmentary abnormalities of the skin. In most Griscelli patients, the RAB27A gene, which encodes a small GTPase that is associated with the melanosome membrane in melanocytes, is mutated. Here we discuss a genomic RAB27A deletion found in a 21-month-old Moroccan Griscelli patient. Additionally, we provide evidence that the loss of functional Rab27a in melanocytes of this Griscelli patient is partially compensated by the up-regulation of Rab27b, a homologue of Rab27a. By real-time quantitative PCR and western blot analysis, we found that Rab27b mRNA and protein, expressed at low levels in normal human melanocytes, is significantly up-regulated in melanocytes derived from this patient. Our immunofluorescence and yeast two-hybrid screening studies reveal that Rab27b can form a tripartite complex on the melanosome membrane with Melanophilin, a Rab27a effector, and protein products of Myosin Va transcripts that contain exon F. Our data suggest that up-regulated Rab27b in melanocytes of the Griscelli patient can partially take over the function of Rab27a, which could explain the fact that this patient had an evenly pigmented skin and was able to tan.

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel trade mark when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-beta (TGF-beta) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-beta. Functional N-cadherin activity is implicated in TGF-beta stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-beta1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-beta stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.

Primary human epidermal melanocytes express six endogenous isoforms of the human actin-associated myosin Va motor protein, involved in organelle transport. As isoforms containing exon F are most abundant in melanocytes, we hypothesized that these isoforms probably have a melanocyte-specific function. To uncover the biologic role of the six isoforms we introduced enhanced green fluorescent protein (eGFP)-myosin Va tail constructs in human melanocytes. We found that the medial tail, undergoing alternative splicing, has to be expressed in combination with the globular tail in order to obtain clear colocalization with organelles. Our data show that isoforms lacking exon F but containing exon D are associated with vesicles near the Golgi area. Myosin Va isoforms containing exon F are able to colocalize with and influence melanosome distribution by indirect interaction with rab27a and direct interaction with melanophilin. These results indicate that the myosin Va medial tail domain provides the globular tail domain with organelle-interacting specificity.

One essential part of the process of skin pigmentation comprises the production of melanosomes, the melanin-containing organelles, and correct transport towards their target cells, the keratinocytes. In this overview the molecular mechanisms of these processes are discussed in view of a number of pigmentation syndromes.

In this review an overview of recent advances in the understanding of melanosome movement within epidermal melanocytes is given. Exploration of the molecular events involved in and determining the process of melanosome transport, as an essential part of human pigmentation, could lead to the identification of agents that augment, or down-regulate the transfer of melanosomes to surrounding keratinocytes. This would present a major breakthrough in the possibilities to influence pigmentation and related disorders, of great concern to some patients. Moreover, melanosome transport offers a good model to study mammalian organelle trafficking and its key players in general.

Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.