Right ventricular overloading is attenuated in monocrotaline-induced pulmonary hypertension model rats with a disrupted Gpr143 gene, the gene that encodes the 3,4-l-dihydroxyphenyalanine (l-DOPA) receptor.

Pulmonary hypertension (PH) is a severe and progressive disease that causes elevated right ventricular systolic pressure, right ventricular hypertrophy and ultimately right heart failure. However, the underlying pathophysiologic mechanisms are poorly understood. We previously showed that 3,4-l-dihydroxylphenyalanine (DOPA) sensitizes vasomotor response to sympathetic tone via coupling between the adrenergic receptor alpha1 (ADRA1) and a G protein-coupled receptor 143 (GPR143), a DOPA receptor. We investigated whether DOPA similarly enhances ADRA1-mediated contraction in pulmonary arteries isolated from rats, and whether GPR143 is involved in the PH pathogenesis. Pretreating the isolated pulmonary arteries with DOPA 1 µM enhanced vasoconstriction in response to phenylephrine, an ADRA1 agonist, but not to U-46619, a thromboxane A2 agonist or endothelin-1. We generated Gpr143 gene-deficient (Gpr143-/y) rats, and confirmed that DOPA did not augment phenylephrine-induced contractile response in Gpr143-/y rat pulmonary arteries. We utilized a rat model of monocrotaline (MCT)-induced PH. In the MCT model, the right ventricular systolic pressure was attenuated in the Gpr143-/y rats than in WT rats. Phenylephrine-induced cell migration and proliferation were also suppressed in Gpr143-/y pulmonary artery smooth muscle cells than in WT cells. Our result suggests that GPR143 is involved in the PH pathogenesis in the rat models of PH.

Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules.

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Human antigen R-mediated mRNA stabilization is required for ultraviolet B-induced autoinduction of amphiregulin in keratinocytes.

All members of the EGF family are produced as transmembrane precursors that are proteolytically processed into soluble forms by disintegrin and metalloproteinases (ADAMs) for autocrine/paracrine pathways. In turn, the ligand-activated EGF receptor (EGFR) induces the expression of EGF family members, so-called "autoinduction." However, it is not well understood how this autoinduction occurs. In this study, we investigated the molecular mechanism of the autoinduction of amphiregulin (AREG), a member of the EGF family. We found that ultraviolet B (UVB) exposure increased the AREG mRNA level by stabilization of its mRNA in a human immortalized keratinocyte cell line, HaCaT. The 3' UTR of AREG mRNA was responsible for binding to an mRNA-binding protein, human antigen R (HuR), and the interaction between AREG mRNA and HuR was enhanced by UVB. Inducible knockdown of HuR expression significantly decreased AREG mRNA stability. Interestingly, treatment of HaCaT cells with an EGFR inhibitor, an EGFR neutralizing antibody, or an ADAM inhibitor destabilized AREG mRNA. In the case of ADAM inhibition, administration of soluble AREG restored the mRNA level, indicating that the stabilization occurs in a shedding-dependent manner of EGFR ligands. The HuR dependence of AREG mRNA and protein expression was also confirmed in human primary keratinocytes. Taken together, we propose a novel mechanism by which HuR regulates the stability of AREG mRNA in keratinocytes after UVB exposure and suggest that targeting of HuR functions might be crucial for understanding skin cancers caused by aberrant EGF family member-EGFR signaling.

Junctional Rab13-binding protein (JRAB) regulates cell spreading via filamins.

We previously showed that Rab13 and its effector protein, junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively 'closed' state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively 'open' state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB-induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA-JRABΔCC, filamin, an actin cross-linking protein, coimmunoprecipitated with HA-JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC-induced cell spreading. Consistent with our previous results, actinin-1/-4 were also immunoprecipitated with HA-JRABΔCC. However, actinin-1/-4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.

Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.

Doxycycline-dependent inducible and reversible RNA interference mediated by a single lentivirus vector.

RNA interference has been applied to the development of a method of silencing genes of interest. Short hairpin RNA (shRNA) expression vectors are useful in driving gene-silencing. Standard shRNA vectors produce a knockdown phenotype soon after transduction. An alternative strategy for conditional gene knockdown would be useful to investigate gene functions in a time-dependent manner. In this study, we developed an inducible gene knockdown system based on lentivirus-mediated gene transfer. A single lentivirus vector capable of inducible expression of a designed microRNA-based shRNA was generated using a tetracycline-dependent transactivation system. The lentiviral vector facilitated doxycycline-dependent inducible knockdown specific to the target gene. Withdrawal of doxycycline after transient treatment resulted in complete recovery of target gene expressions to normal levels. Thus the single lentiviral vector developed in this study should be a powerful tool for doxycycline-dependent inducible and reversible RNA interference in molecular genetic studies.

Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents.

We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Reward expectation enhances action-related activity of nigral dopaminergic and two striatal output pathways.

Neurons comprising nigrostriatal system play important roles in action selection. However, it remains unclear how this system integrates recent outcome information with current action (movement) and outcome (reward or no reward) information to achieve appropriate subsequent action. We examined how neuronal activity of substantia nigra pars compacta (SNc) and dorsal striatum reflects the level of reward expectation from recent outcomes in rats performing a reward-based choice task. Movement-related activity of direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) were enhanced by reward expectation, similarly to the SNc dopaminergic neurons, in both medial and lateral nigrostriatal projections. Given the classical basal ganglia model wherein dopamine stimulates dSPNs and suppresses iSPNs through distinct dopamine receptors, dopamine might not be the primary driver of iSPN activity increasing following higher reward expectation. In contrast, outcome-related activity was affected by reward expectation in line with the classical model and reinforcement learning theory, suggesting purposive effects of reward expectation.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

Role of activation-induced cytidine deaminase in the progression of follicular lymphoma.

Activation-induced cytidine deaminase (AID/AICDA) is required for somatic hypermutation and class-switch recombination of the immunoglobulin gene, and for c-myc translocation of germinal center-derived B-cell lymphoma. In the present study, we attempted to clarify the significance of AID associated with c-myc in the progression of follicular lymphoma (FL) using RT-PCR and quantitative real-time PCR. Tissues from the patients with grade 3 FL expressed relatively higher levels of c-myc and AID. The samples taken from a patient with FL who died within 2 years after the start of treatment showed either no or low expression of AID, despite expressing high levels of c-myc. In order to examine the role of AID expression in rapidly progressive FL, the full-length AID transcript was transfected into AID-negative cell lines established from different patients with rapidly progressive FL. This led to the establishment of AID-expressing transfectants with a low proliferation rate and a significantly increased incidence of G(0)/G(1) arrest compared with controls. Our results indicate that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may correlate with the clinical outcomes of FL.

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology.

BACKGROUND: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. NEW METHOD: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. RESULTS: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. CONCLUSIONS: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.

PURPOSE: To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line. METHODS: iHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs. RESULTS: The iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days. CONCLUSION: The iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.

Membrane-anchored growth factors, the epidermal growth factor family: beyond receptor ligands.

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane-anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross-talk in response to various shedding stimulants such as G-protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR-ligands, heparin-binding EGF-like growth factor (HB-EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB-EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy-terminal fragments to regulate gene expression directly.

Conformational plasticity of JRAB/MICAL-L2 provides "law and order" in collective cell migration.

In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration.

Conditional ablation of striatal neuronal types containing dopamine D2 receptor disturbs coordination of basal ganglia function.

Dopamine (DA) exerts synaptic organization of basal ganglia circuitry through a variety of neuronal populations in the striatum. We performed conditional ablation of striatal neuronal types containing DA D2 receptor (D2R) by using immunotoxin-mediated cell targeting. Mutant mice were generated that express the human interleukin-2 receptor alpha-subunit under the control of the D2R gene. Intrastriatal immunotoxin treatment of the mutants eliminated the majority of the striatopallidal medium spiny neurons and cholinergic interneurons. The elimination of these neurons caused hyperactivity of spontaneous movement and reduced motor activation in response to DA stimulation. The elimination also induced upregulation of GAD gene expression in the globus pallidus (GP) and downregulation of cytochrome oxidase activity in the subthalamic nucleus (STN), whereas it attenuated DA-induced expression of the immediate-early genes (IEGs) in the striatonigral neurons. In addition, chemical lesion of cholinergic interneurons did not alter spontaneous movement but caused a moderate enhancement in DA-induced motor activation. This enhancement of the behavior was accompanied by an increase in the IEG expression in the striatonigral neurons. These data suggest that ablation of the striatopallidal neurons causes spontaneous hyperactivity through modulation of the GP and STN activity and that the ablation leads to the reduction in DA-induced behavior at least partly through attenuation of the striatonigral activity as opposed to the influence of cholinergic cell lesion. We propose a possible model in which the striatopallidal neurons dually regulate motor behavior dependent on the state of DA transmission through coordination of the basal ganglia circuitry.

Survival of developing motor neurons mediated by Rho GTPase signaling pathway through Rho-kinase.

A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPase Rho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine-threonine protein kinase (Rho-kinase-ROK-ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA (RhoA DN) or for Rho-kinase (Rho-K DN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DN showed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.

Arkadia enhances BMP signalling through ubiquitylation and degradation of Smad6.

Arkadia, a positive regulator of Smad-dependent signalling via the transforming growth factor-ß (TGF-ß) family, is an E3 ubiquitin ligase that induces ubiquitylation and proteasome-dependent degradation of TGF-ß suppressors such as Smad7, c-Ski and SnoN. In this study, we examined the effects of Arkadia on bone morphogenetic protein (BMP)-induced osteoblast differentiation. Knockdown of Arkadia reduced mineralization and expression of osteoblast differentiation markers. Furthermore, we showed that Smad6, a BMP-specific inhibitory Smad, is a target of Arkadia: wild-type (WT) Arkadia, but not the C937A (CA) mutant lacking E3 ubiquitin-ligase activity, induced ubiquitylation and proteasome-dependent degradation of Smad6. Accordingly, protein levels of Smad6, Smad7 and c-Ski were elevated in MEFs from Arkadia KO mice. Finally, expression of Arkadia attenuated blockade of BMP signalling by Smad6 in a transcriptional reporter assay. These results demonstrate that Smad6 is a novel target of Arkadia, and that Arkadia positively regulates BMP signalling via degradation of Smad6, Smad7 and c-Ski/SnoN.

Rotation is the primary motion of paired human epidermal keratinocytes.

BACKGROUND: Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. OBJECTIVE: The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. METHODS: We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. RESULTS: The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. CONCLUSION: The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis.