PI 3-Kinase and the Histone Methyl-Transferase KMT2D Collaborate to Induce Arp2/3-Dependent Migration of Mammary Epithelial Cells.

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.

Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration.

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

Divergent Contribution of the Golgi Apparatus to Microtubule Organization in Related Cell Lines.

Membrane trafficking in interphase animal cells is accomplished mostly along the microtubules. Microtubules are often organized radially by the microtubule-organizing center to coordinate intracellular transport. Along with the centrosome, the Golgi often serves as a microtubule-organizing center, capable of nucleating and retaining microtubules. Recent studies revealed the role of a special subset of Golgi-derived microtubules, which facilitates vesicular traffic from this central transport hub of the cell. However, proteins essential for microtubule organization onto the Golgi might be differentially expressed in different cell lines, while many potential participants remain undiscovered. In the current work, we analyzed the involvement of the Golgi complex in microtubule organization in related cell lines. We studied two cell lines, both originating from green monkey renal epithelium, and found that they relied either on the centrosome or on the Golgi as a main microtubule-organizing center. We demonstrated that the difference in their Golgi microtubule-organizing activity was not associated with the well-studied proteins, such as CAMSAP3, CLASP2, GCC185, and GMAP210, but revealed several potential candidates involved in this process.

Synthesis, Screening and Characterization of Novel Potent Arp2/3 Inhibitory Compounds Analogous to CK-666.

Branched actin networks polymerized by the Actin-related protein 2 and 3 (Arp2/3) complex play key roles in force generation and membrane remodeling. These networks are particularly important for cell migration, where they drive membrane protrusions of lamellipodia. Several Arp2/3 inhibitory compounds have been identified. Among them, the most widely used is CK-666 (2-Fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide), whose mode of action is to prevent Arp2/3 from reaching its active conformation. Here 74 compounds structurally related to CK-666 were screened using a variety of assays. The primary screen involved EdU (5-ethynyl-2'-deoxyuridine) incorporation in untransformed MCF10A cells. The resulting nine positive hits were all blocking lamellipodial protrusions and cell migration in B16-F1 melanoma cells in secondary screens, showing that cell cycle progression can be a useful read-out of Arp2/3 activity. Selected compounds were also characterized on sea urchin embryos, where Arp2/3 inhibition yields specific phenotypes such as the lack of triradiate spicules and inhibition of archenteron elongation. Several compounds were filtered out due to their toxicity in cell cultures or on sea urchin development. Two CK-666 analogs, 59 (N-{2-[5-(Benzyloxy)-2-methyl-1H-indol-3-yl] ethyl}-3-bromobenzamide) and 69 (2,4-Dichloro-N-[2-(7-chloro-2-methyl-1H-indol-3-yl) ethyl]-5-[(dimethylamino) sulfonyl] benzamide), were active in all assays and significantly more efficient in vivo than CK-666. These best hits with increased in vivo potency were, however, slightly less efficient in vitro than CK-666 in the classical pyrene-actin assay. Induced-fit docking of selected compounds and their possible metabolites revealed interaction with Arp2/3 that suppresses Arp2/3 activation. The data obtained in our screening validated the applicability of original assays for Arp2/3 activity. Several previously unexplored CK-666 structural analogs were found to suppress Arp2/3 activation, and two of them were identified as Arp2/3 inhibitors with improved in vivo efficiency.

The Role of the Adapter Protein Anks1a in the Regulation of Breast Cancer Cell Motility.

Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression that leads to the acquisition by cancer cells the capacity for migration using the mesenchymal motility mode regulated by the Rac→WAVE→Arp2/3 signaling pathway. Earlier it was shown that proteins interacting with Rac can regulate mesenchymal migration and thus determine the metastatic potential of the cells. The search for new regulators of cell migration is an important theoretical and practical task. The adaptor protein Anks1a is one of the proteins interacting with Rac, whose expression is altered in many types of tumors. The aim of this study was to find whether Anks1a affects the migration of cancer cells and to identify the mechanism underlying this effect. It was suggested that Anks1a can influence cancer cell migration either as a Rac1 effector or by activating human epidermal growth factor receptor 2 (HER2) exchange. We investigated how upregulation and inhibition of Anks1a expression affected migration of breast cancer cells with different HER2 status. Anks1a was shown to interact with the activated form of Rac1. In the MDA-MB-231 cells (triple negative cancer), which lack HER2, Anks1a accumulated at the active cell edge, which is characterized by enrichment with active Rac1, whereas no such accumulation was observed in the HER2-overexpressing SK-BR-3 cells. Downregulation of the ANKS1a expression with esiRNA had almost no effect on the cancer cell motility, except a slight increase in the average migration rate of MDA-MB-231 cells. Among three cell lines tested, overexpression of Anks1a increased the migration rate of HER2-overexpressng SK-BR-3 cells only. We showed that Anks1a is an effector of activated Rac1, but its influence on the cell migration in this capacity was minimal, at least in the studied breast cancer cells. Anks1a affected the motility of breast cancer cells due to its involvement in the EGF receptor exchange.

Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration.

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.

Assembly and Activity of the WASH Molecular Machine: Distinctive Features at the Crossroads of the Actin and Microtubule Cytoskeletons.

The Arp2/3 complex generates branched actin networks at different locations of the cell. The WASH and WAVE Nucleation Promoting Factors (NPFs) activate the Arp2/3 complex at the surface of endosomes or at the cell cortex, respectively. In this review, we will discuss how these two NPFs are controlled within distinct, yet related, multiprotein complexes. These complexes are not spontaneously assembled around WASH and WAVE, but require cellular assembly factors. The centrosome, which nucleates microtubules and branched actin, appears to be a privileged site for WASH complex assembly. The actin and microtubule cytoskeletons are both responsible for endosome shape and membrane remodeling. Motors, such as dynein, pull endosomes and extend membrane tubules along microtubule tracks, whereas branched actin pushes onto the endosomal membrane. It was recently uncovered that WASH assembles a super complex with dynactin, the major dynein activator, where the Capping Protein (CP) is exchanged from dynactin to the WASH complex. This CP swap initiates the first actin filament that primes the autocatalytic nucleation of branched actin at the surface of endosomes. Possible coordination between pushing and pulling forces in the remodeling of endosomal membranes is discussed.

Arpin Regulates Migration Persistence by Interacting with Both Tankyrases and the Arp2/3 Complex.

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid-liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.

The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex.

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

Centrosome-derived microtubule radial array, PCM-1 protein, and primary cilia formation.

In animal cells, the centrosome nucleates and anchors microtubules (MT), forming their radial array. During interphase centrosome-derived MT, aster can either team up with other MT network or function in an autonomous manner. What is the function of the centrosome-derived MT aster? We suggested that it might play an important role in the formation of the primary cilium, the organelle obligatorily associated with the centrosome. PCM-1 (PeriCentriolar Matrix 1) protein, which participates in the organization of the primary cilium, is a part of pericentiolar satellites. They are transported to the centrosome along MTs by the motor protein dynein in a complex with its cofactor dynactin. Previously, we showed that SLK/LOSK phosphorylated the p150Glued subunit of dynactin, thus promoting its centrosomal targeting followed by its participation in the retention of microtubules. Here, we found that under the repression of SLK/LOSK activity, the PCM-1 protein lost its pericentrosomal localization and was being dispersed throughout the cytoplasm. Despite that the alanine and glutamine mutants of p150Glued had opposite effects on PCM-1 localization, they associated with PCM-1 to the same extent. The occurrence of primary cilia also significantly decreased when SLK/LOSK was repressed. These defects also correlated with a disturbance of the long-range transport in cells, whereas dynein-depending motility was intact. Treatment with the GSK-3ß kinase inhibitor also resulted in the loss of the centrosome-derived MT aster, dispersion of PCM-1 over the cytoplasm, and reduction of primary cilia occurrence. Thus, kinases involved in the centrosome-derived MT aster regulation can indirectly control the formation of primary cilia in cells.

MAST-like protein kinase IREH1 from Arabidopsis thaliana co-localizes with the centrosome when expressed in animal cells.

MAIN CONCLUSION: The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well.

SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization.

Cell motility is an essential complex process that requires actin and microtubule cytoskeleton reorganization and polarization. Such extensive rearrangement is closely related to cell polarization as a whole. The serine/threonine kinase SLK/LOSK is a potential regulator of cell motility, as it phosphorylates a series of cytoskeleton-bound proteins that collectively participate in the remodeling of migratory cell architecture. In this work, we report that SLK/LOSK is an indispensable regulator of cell locomotion that primarily acts through the small GTPase RhoA and the dynactin subunit p150(Glued). Both RhoA and dynactin affect cytoskeleton organization, polarization, and general cell locomotory activity to various extents. However, it seems that these events are independent of each other. Thus, SLK/LOSK kinase effectively functions as a switch that links all of the processes underlying cell motility to provide robust directional movement.

Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome.

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end-directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150(Glued) subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150(Glued) and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150(Glued) to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150(Glued) expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150(Glued) suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.