Extracellular vesicles released by keratinocytes regulate melanosome maturation, melanocyte dendricity, and pigment transfer.

Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids, and genetic material between cells and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthesize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes [(A. Lo Cicero et al., Nat. Commun. 6, 7506 (2015)]. However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here, we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites and mature melanosomes and promote their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulating pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors to cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.

Plekhh1, a partner of myosin 1 and an effector of EphB2, controls the cortical actin network during cell repulsion.

EphB2-ephrinB signalling, which plays a major role in cell segregation during embryonic development and tissue homeostasis, induces an important reorganization of the cortical actin network. We have previously reported that myosin 1b contributes to reorganization of the cortical actin network upon EphB2 signalling. In this report, we identify Plekhh1 as a new partner of members of the myosin 1 family and EphB2 receptors. Plekhh1 interacts with myosin 1b via its N-terminal domain and with EphB2 via its C-terminal domain. Furthermore, Plekhh1 is tyrosine phosphorylated, and this depends on EphB2 kinase activity. Similar to the effects of manipulating levels of myosin 1b and myosin 1c, manipulation of Plekhh1 expression levels alters the formation of filopodia, the length of focal adhesions and the formation of blebs. Furthermore, binding of the Plekhh1 interacting domain to myosin 1b increases the motor activity of myosin 1b in vitro. Taken together, our data show that Plekhh1 is an effector of EphB2 and suggest that Plekhh1 regulates the cortical actin network via the interaction of its N-terminal domain with myosin 1 upon EphB2-ephrinB signalling.

Myosin 1b promotes axon formation by regulating actin wave propagation and growth cone dynamics.

Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking. We show that Myo1b controls the dynamics of the growth cones and the anterograde propagation of the actin waves. By coupling the membrane to the actin cytoskeleton, Myo1b regulates the size of the actin network as well as the stability and size of filopodia in the growth cones. Our data provide the first evidence that a myosin 1 plays a major role in neuronal symmetry breaking and argue for a mechanical control of the actin cytoskeleton both in actin waves and in the growth cones by this myosin.

Myosin 1b functions as an effector of EphB signaling to control cell repulsion.

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.

Cloning of the mRNA of overexpression in colon carcinoma-1: a sequence overexpressed in a subset of colon carcinomas.

We have identified a novel human cDNA overexpressed in a colon carcinoma cell line, TC7, established from a tumor with a normal karyotype arising in a patient with a hereditary nonpolyposis colorectal carcinoma. The OCC-1 (overexpressed in colon carcinoma-1) gene is composed of six exons and located in the q24.1 region of chromosome 12. It is transcribed as two mRNA species that differ in their 5'- and 3'-terminal ends. Abundant accumulation of both transcripts was found in placenta, skeletal muscle, kidney, and pancreas tissues. Absent or very faint expression was observed in heart, brain, lung and liver tissues. Overexpressed in colon carcinoma-1 cDNA direct in vitro translation of several polypeptides whose size is shorter than 9 kDa. Attempts to produce antibodies against these synthesized polypeptides in Escherichia coli failed. The absence of sequences at the mRNA and DNA levels hybridizing with mouse sequences together with the absence of a large open reading frame raise the possibility that OCC-1 sequences appeared recently in evolution and are transcribed as two noncoding regulatory RNA. Elevated levels of OCC-1 mRNA were observed in three of eight colon carcinomas as compared to normal mucosa of the same patient. Since these tumors shared the same characteristics of having a near diploid karyotype, OCC-1 overexpression may be a hallmark of this subset of colon carcinomas.

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.

Zinc finger protein overexpressed in colon carcinoma interacts with the telomeric protein hRap1.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We found an interaction between OZF and the telomeric hRap1 protein with a yeast two-hybrid screen. hRap1 (TERF2IP) is an ortholog of the yeast telomeric protein, scRap1 originally identified as a regulator of telomere length. In HeLa cells, it interacts with TRF2, a telomere repeat binding factor whose inactivation causes a dysregulation of telomere length and structure. Immunoprecipitation with anti-hRap1 antibodies in conditions that allow the purification of proteins associated with hRap1, demonstrated that OZF binds to hRap1 in HeLa cells. Using deletion mutants, we mapped the interacting domain of each protein. The three zinc fingers at the C-terminus of OZF interact with a region of hRap1 located downstream of the coil domain. It involves a stretch of at least 25 amino acids at the C-terminus of hRap1 that interact with TRF2. This suggests that OZF overexpression in tumours may alter the balance between hRap1 and other telomeric proteins and therefore that OZF function may be linked to telomere regulation.