MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.

Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs, often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown. Here, we investigated the regulators of MVB fate by analyzing the effects of signaling inhibitors on EV secretion from cancer cells engineered to secrete luciferase-labeled EVs. Inhibition of the oncogenic MEK/ERK pathway suppressed EV release and activated lysosome formation. MEK/ERK-mediated lysosomal inactivation impaired MVB degradation, resulting in increased EV secretion from cancer cells. Moreover, MEK/ERK inhibition prevented c-MYC expression and induced the nuclear translocation of MiT/TFE transcription factors, thereby promoting the activation of lysosome-related genes, including the gene encoding a subunit of vacuolar-type H+ -ATPase, which is responsible for lysosomal acidification and function. Furthermore, c-MYC upregulation was associated with lysosomal gene downregulation in MEK/ERK-activated renal cancer cells/tissues. These findings suggest that the MEK/ERK/c-MYC pathway controls MVB fate and promotes EV production in human cancers by inactivating lysosomal function.

Propagating Wave of ERK Activation Orients Collective Cell Migration.

The biophysical framework of collective cell migration has been extensively investigated in recent years; however, it remains elusive how chemical inputs from neighboring cells are integrated to coordinate the collective movement. Here, we provide evidence that propagation waves of extracellular signal-related kinase (ERK) mitogen-activated protein kinase activation determine the direction of the collective cell migration. A wound-healing assay of Mardin-Darby canine kidney (MDCK) epithelial cells revealed two distinct types of ERK activation wave, a "tidal wave" from the wound, and a self-organized "spontaneous wave" in regions distant from the wound. In both cases, MDCK cells collectively migrated against the direction of the ERK activation wave. The inhibition of ERK activation propagation suppressed collective cell migration. An ERK activation wave spatiotemporally controlled actomyosin contraction and cell density. Furthermore, an optogenetic ERK activation wave reproduced the collective cell migration. These data provide new mechanistic insight into how cells sense the direction of collective cell migration.

Age-dependent deterioration of locomotion in Drosophila melanogaster deficient in the homologue of amyotrophic lateral sclerosis 2.

Recessive mutations in the amyotrophic lateral sclerosis 2 (ALS2) gene have been linked to juvenile-onset ALS2. Although one of the molecular functions of the ALS2 protein is clearly the activation of Rab5, the mechanisms underlying the selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood. Here, we focused on the ALS2 homologue of Drosophila melanogaster, isolated two independent deletions, and systematically compared phenotypes of the mutants with those of animals in which Rab5 function in identified neurons was abrogated. In the dALS2 mutant flies, we found that the stereotypic axonal and dendritic morphologies of neurons shared some features with those in Rab5-deficient flies, but the dALS2 mutant phenotypes were much milder. We also found that the abrogation of Rab5 function in motor neurons strongly depressed the locomotion activity of adults, resembling the behavior of aged dALS2 mutants. Importantly, this age-dependent locomotion deficit of dALS2 mutants was restored to normal by expressing the dALS2 transgene in a wide range of tissues. This finding provided a platform where we could potentially identify particular cell types responsible for the phenotype by tissue-specific rescue experiments. We discuss our results and the future usage of the dALS2 mutant as a new ALS model.

Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF.

Rac1 has a crucial role in epidermal growth factor (EGF)-induced membrane ruffling, lamellipodial protrusion, and cell migration. Several guanine nucleotide exchange factors (GEFs) including Sos1, Sos2, Tiam1 and Vav2 have been shown to transduce the growth signal from the EGF receptor to Rac1. To clarify the role of each GEF, we time-lapse imaged the EGF-induced activity change of Rac1 in A431 cells transfected with siRNA targeting each Rac1 GEF. Because knockdown of these GEFs suppressed EGF-induced Rac1 activation only partially, we looked for another Rac1 GEF downstream of the EGF receptor and found that Asef, a Rac1-Cdc42 GEF bound to the tumor suppressor APC, also contributed to EGF-induced Rac1 activation. Intriguingly, EGF stimulation induced phosphorylation of Tyr94 within the APC-binding region of Asef in a manner dependent on Src-family tyrosine kinases. The suppression of EGF-induced Rac1 activation in siRNA-treated cells was restored by wild-type Asef, but not by the Tyr94Phe mutant of Asef. This observation strongly argues for the positive role of Tyr94 phosphorylation in EGF-induced Asef activation following the activation of Rac1.

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src.

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.

Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes.

To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.

A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes.

Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.

Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor.

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells.

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.