Characterizing the Molecular Mechanism of the Lethal C423D Mutation in FgMyoI: A Molecular Perspective.

The lethal mutation C423D in Fusarium graminearum myosin I (FgMyoI) occurs close to the binding pocket of the allosteric inhibitor phenamacril and causes severe inhibition on mycelial growth of F. graminearum strain PH-1. Here, based on extensive Gaussian accelerated molecular dynamics simulations and wet experiments, we elucidate the underlying molecular mechanism of the abnormal functioning of the FgMyoIC423D mutant at the atomistic level. Our results suggest that the damaging mutation C423D exhibits a synergistic allosteric inhibition mechanism similar to but more robust than that of phenamacril, including effects on the active site and actin binding. Unlike phenamacril-induced closure of Switch2, the mutation results in unfolding of the N-terminal relay helix with a partially opened Switch2 and blocks the structural rearrangement of the relay/SH1 helices, impairing the proper initiation of the recovery stroke. Due to the significant influence of C423D mutation on the function of FgMyoI, designing covalent inhibitors targeting this site holds tremendous potential.

RAB31 in glioma-derived endothelial cells promotes glioma cell invasion via extracellular vesicle-mediated enrichment of MYO1C.

Extracellular vesicles (EV), important messengers in intercellular communication, can load and transport various bioactive components and participate in different biological processes. We previously isolated glioma human endothelial cells (GhECs) and found that GhECs, rather than normal human brain endothelial cells (NhECs), exhibit specific enrichment of MYO1C into EVs and promote the migration of glioma cells. In this study, we explored the mechanism by which MYO1C is secreted into EVs. We report that such secretion is dependent on RAB31, RAB27B, and FAS. When expression of RAB31 increases, MYO1C is enriched in secretory EVs. Finally, we identified an EV export mechanism for MYO1C that promotes glioma cell invasion and is dependent on RAB31 in GhECs. In summary, our data indicate that the knockdown of RAB31 can reduce enrichment of MYO1C in extracellular vesicles, thereby attenuating the promotion of glioma cell invasion by GhEC-EVs.

Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis.

Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.

Endocytic myosin-1 is a force-insensitive, power-generating motor.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

CCND1-associated ceRNA network reveal the critical pathway of TPRG1-AS1-hsa-miR-363-3p-MYO1B as a prognostic marker for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer death globally, yet there are few useful biomarkers for early identification and prognostic prediction. Previous studies have confirmed that CCND1 amplification is closely associated with head and neck oncogenesis, and the present study explored the ceRNA network associated with CCND1. Gene expression profiling of the Head and Neck Squamous Cell Carcinoma (HNSC) project of The Cancer Genome Atlas (TCGA) program identified the TPRG1-AS1-hsa-miR-363-3P-MYO1B gene regulatory axis associated with CCND1. Further analysis of the database showed that MYOB was regulated by methylation in head and neck tumors, and functional enrichment analysis showed that MYO1B was involved in "actin filament organization" and "cadherin binding ". Immune infiltration analysis suggested that MYO1B may influence tumorigenesis and prognosis by regulating the immune microenvironment of HNSC. MYO1B enhanced tumor spread through the EMT approach, according to epithelial mesenchymal transition (EMT) characterisation. We analyzed both herbal and GSCALite databases and found that CCND1 and MYO1B have the potential as predictive biomarkers for the treatment of HNSC patients. RT-qPCR validated bioinformatic predictions of gene expression in vitro cell lines. In conclusion, we found a CCND1-related ceRNA network and identified the novel TPRG1-AS1-hsa-miR-363-3p-MYO1B pathway as a possible HNSC diagnostic biomarker and therapeutic target.

Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics.

Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in in vitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in in vitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.

hucMSCs treatment prevents pulmonary fibrosis by reducing circANKRD42-YAP1-mediated mechanical stiffness.

Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial pneumonia of unknown cause. The most typical characteristic of IPF is gradual weakening of pulmonary elasticity and increase in hardness/rigidity with aging. This study aims to identify a novel treatment approach for IPF and explore mechanism of mechanical stiffness underlying human umbilical cord mesenchymal stem cells (hucMSCs) therapy. Target ability of hucMSCs was examined by labeling with cell membrane dye Dil. Anti-pulmonary fibrosis effect of hucMSCs therapy by reducing mechanical stiffness was evaluated by lung function analysis and MicroCT imaging system and atomic force microscope in vivo and in vitro. Results showed that stiff environment of fibrogenesis caused cells to establish a mechanical connection between cytoplasm and nucleus, initiating expression of related mechanical genes such as Myo1c and F-actin. HucMSCs treatment blocked force transmission and reduced mechanical force. For further exploration of mechanism, ATGGAG was mutated to CTTGCG (the binding site of miR-136-5p) in the full-length sequence of circANKRD42. Wildtype and mutant plasmids of circANKRD42 were packaged into adenovirus vectors and sprayed into lungs of mice. Mechanistic dissection revealed that hucMSCs treatment repressed circANKRD42 reverse splicing biogenesis by inhibiting hnRNP L, which in turn promoted miR-136-5p binds to 3'-Untranslated Region (3'-UTR) of YAP1 mRNA directly, thus inhibiting translation of YAP1 and reducing YAP1 protein entering nucleus. The condition repressed expression of related mechanical genes to block force transmission and reduce mechanical forces. The mechanosensing mechanism mediated directly by circANKRD42-YAP1 axis in hucMSCs treatment, which has potential general applicability in IPF treatment.

Myo1f has an essential role in γδT intraepithelial lymphocyte adhesion and migration.

γδT intraepithelial lymphocyte represents up to 60% of the small intestine intraepithelial compartment. They are highly migrating cells and constantly interact with the epithelial cell layer and lamina propria cells. This migratory phenotype is related to the homeostasis of the small intestine, the control of bacterial and parasitic infections, and the epithelial shedding induced by LPS. Here, we demonstrate that Myo1f participates in the adhesion and migration of intraepithelial lymphocytes. Using long-tailed class I myosins KO mice, we identified the requirement of Myo1f for their migration to the small intestine intraepithelial compartment. The absence of Myo1f affects intraepithelial lymphocytes' homing due to reduced CCR9 and α4ß7 surface expression. In vitro, we confirm that adhesion to integrin ligands and CCL25-dependent and independent migration of intraepithelial lymphocytes are Myo1f-dependent. Mechanistically, Myo1f deficiency prevents correct chemokine receptor and integrin polarization, leading to reduced tyrosine phosphorylation which could impact in signal transduction. Overall, we demonstrate that Myo1f has an essential role in the adhesion and migration in γδT intraepithelial lymphocytes.

Characterizing the molecular impact of KMT2D variants on the epigenetic and transcriptional landscapes in Kabuki syndrome.

Kabuki syndrome (KS) is a rare, multisystem disorder with a variable clinical phenotype. The majority of KS is caused by dominant loss-of-function mutations in KMT2D (lysine methyltransferase 2D). KMT2D mediates chromatin accessibility by adding methyl groups to lysine residue 4 of histone 3, which plays a critical role in cell differentiation and homeostasis. The molecular underpinnings of KS remain elusive partly because of a lack of histone modification data from human samples. Consequently, we profiled and characterized alterations in histone modification and gene transcription in peripheral blood mononuclear cells (PBMCs) from 33 patients with KMT2D mutations and 36 unaffected healthy controls. Our analysis identified unique enhancer signatures in H3K4me1 and H3K4me2 in KS compared with controls. Reduced enhancer signals were present for promoter-distal sites of immune-related genes for which co-binding of PBMC-specific transcription factors was predicted; 31% of super-enhancers of normal blood cells overlapped with disrupted enhancers in KS, supporting an association of reduced enhancer activity of immune-related genes with immune deficiency phenotypes. In contrast, increased enhancer signals were observed for promoter-proximal regions of metabolic genes enriched with EGR1 and E2F2 motifs, whose transcriptional levels were significantly increased in KS. Additionally, we identified ~100 de novo enhancers in genes, such as in MYO1F and AGAP2. Together, our results underscore the effect of KMT2D haploinsufficiency on dysregulation of enhancer states and gene transcription and provide a framework for the identification of therapeutic targets and biomarkers in preparation for clinical trial readiness.

MYO1B as a prognostic biomarker and a therapeutic target in Arecoline-associated oral carcinoma.

BACKGROUND: Arecoline, the main component of betel nut, induces malignant transformation of oral cells through complicated unclear mechanisms. Thus, we aimed to screen the key genes involved in Arecoline-induced oral cancer and further verify their expressions and roles. METHODS: This study included a data-mining part, a bioinformatics verification part, and an experimental verification one. First, the key gene related to oral cancer induced by Arecoline was screened. Then, the expression and clinical significance of the key gene in head and neck/oral cancer tissues were verified, and its downstream mechanisms of action were explored. Afterwards, the expression and roles of the key gene were verified by experiments at the histological and cytological levels. RESULTS: MYO1B was identified as the key gene. Overexpression of MYO1B was associated with lymph node metastasis and unfavorable outcomes in oral cancer. MYO1B may be mainly related to metastasis, angiogenesis, hypoxia, and differentiation. A positive correlation between MYO1B and the infiltration of macrophages, B cells, and dendritic cells was presented. MYO1B might have a close relationship with SMAD3, which may be enriched in the Wnt signaling pathway. MYO1B suppression markedly inhibited the proliferation, invasion, and metastasis abilities of both Arecoline-transformed oral cells and oral cancer cells. CONCLUSION: This study revealed MYO1B as a key gene in Arecoline-induced oral tumorigenesis. MYO1B might be a novel prognostic indicator and therapeutic target for oral cancer.

Myo1e overexpression in lung adenocarcinoma is associated with increased risk of mortality.

This study aims to perform a comprehensive genomic analysis to assess the influence of overexpression of MYO1E in non-small cell lung carcinoma (NSCLC) and whether there are differences in survival and mortality risk in NSCLC patients depending on both DNA methylation and RNA expression of MYO1E. The DNA methylation probe cg13887966 was inversely correlated with MYO1E RNA expression in both LUAD and LUSC subpopulations showing that lower MYO1E RNA expression was associated with higher MYO1E DNA methylation. Late stages of lung cancer showed significantly lower MYO1E DNA methylation and significantly higher MYO1E RNA expression for LUAD but not for LUSC. Low DNA methylation as well as high RNA expression of MYO1E are associated with a shorter median survival time and an increased risk of mortality for LUAD, but not for LUSC. This study suggests that changes in MYO1E methylation and expression in LUAD patients may have an essential role in lung cancer's pathogenesis. It shows the utility of MYO1E DNA methylation and RNA expression in predicting survival for LUAD patients. Also, given the low normal expression of MYO1E in blood cells MYO1E DNA methylation has the potential to be used as circulating tumor marker in liquid biopsies.

Elevation of truncated (48 kDa) form of unconventional myosin 1C in blood serum correlates with severe Covid-19.

In Covid-19 and autoimmune patients, there are several similarities revealed in the immune responses (Liu et al., 2021; Woodruff et al., 2020). Earlier, we firstly detected a truncated (48 kDa) form of the unconventional Myosin 1C (48/Myo1C) in a fraction of proteins soluble in 10% 2,2,2-trichloroacetic acid (TCA). These proteins were obtained from blood serum of patients with autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis (Kit et al., 2018). Here, we demonstrated that content of 48/Myo1C was also elevated in blood serum of the severe Covid-19 patients. Whereas in blood of 28 clinically healthy human individuals regularly tested for Covid-19 infection, the amount of this protein was undetectable or very low, in blood of 16 of 28 patients hospitalized with severe course of this disease, its amount was significantly increased. Dexamethasone, steroid hormone which is widely used for treatment of severe Covid-19 patients, induced time-dependent elevation of the 48/Myo1C in blood of such patients. The 48/Myo1C dose-dependently suppressed the viability of anti-CD3-activated lymphocytes of human peripheral blood. Recently, we used affinity chromatography on the magnetic poly(glycidyl-methacrylate) (mag-PGMA-NH2) microparticles functionalized with Myo1C and MALDI-TOF mass spectrometry with molecular modeling in silico in order to identify potential molecular partners of the 48/Myo1C. It was found that 48/Myo1C might bind to component 3 of the complement system and the anti-thrombin-III (Starykovych et al., 2021). Thus, the mechanisms of the pathogenic action of truncated form of Myo1C in severe COVID-19 patients may involve a suppression of the immune cells, as well as modulation of complement and coagulation cascades.

m6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner.

Emerging evidence indicates the critical roles of N6-methyladenosine (m6A) modification in human cancers. Herein, our work reported that a novel m6A-modified circRNA from the MYO1C gene, circMYO1C, upregulated in the pancreatic ductal adenocarcinoma (PDAC). Our findings demonstrated that circMYO1C is highly expressed in PDAC tissues. Functionally, circMYO1C promoted the proliferation and migration of PDAC cells in vitro and its silencing reduced the tumor growth in vivo. Mechanistically, circMYO1C cyclization was mediated by m6A methyltransferase METTL3. Moreover, methylated RNA immunoprecipitation sequencing (MeRIP-seq) unveiled the remarkable m6A modification on PD-L1 mRNA. Moreover, circMYO1C targeted the m6A site of PD-L1 mRNA to enhance its stability by cooperating with IGF2BP2, thereby accelerating PDAC immune escape. In conclusion, these findings highlight the oncogenic role of METTL3-induced circMYO1C in PDAC tumorigenesis via an m6A-dependent manner, inspiring a novel strategy to explore PDAC epigenetic therapy.

Myo1b Promotes Premature Endothelial Senescence and Dysfunction via Suppressing Autophagy: Implications for Vascular Aging.

Endothelial cell (EC) senescence characterized by an irreversible growth arrest leading to endothelial dysfunction has been implicated in vascular aging and aging-associated cardiovascular diseases. Autophagy plays a crucial role in the modulation of cellular senescence. Our previous showed that myosin 1b (Myo1b), one family of nonfilamentous class-1 myosin, was reported to be involved in the modulation of human smooth muscle cell senescence. However, the role of Myo1b in the modulation of EC senescence with links to autophagy has yet to be elucidated. In this study, we sought to explore the role of Myo1b in endothelial senescence and further elucidate the underlying mechanisms. Here, we show prominent upregulation of Myo1b in senescent ECs in comparison with nonsenescence ECs in both mRNA and protein expression levels. Silencing Myo1b in senescent cells ameliorates endothelial dysfunctions and reverses endothelial senescence phenotypic changes such as senescence-associated-ß-galactosidase activity, cyclin-dependent kinase inhibitor p21WAF1, expression of vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and the senescence-associated cytokines. In contrast, in nonsenescent cells, overexpressing Myo1b promotes endothelial senescence and suppresses autophagy through the impairment of autophagosome and lysosome fusion. The interaction between Myo1b and LRRK2 through Myo1b tail domain promotes intracellular calcium elevation, which results in the inhibition of autophagic flux. In vitro and in vivo aging models, Myo1b knockdown in senescent ECs and wild type-aged mice is able to enhance autophagy and ameliorate aging-associated endothelial dysfunction. Taken together, our studies reveal a new function for Myo1b, that is, to couple LRRK2 assembly to promote an increase in intracellular calcium level, which impairs the autophagosome-lysosome fusion, and ultimately the promotion of EC senescence and vascular aging.

Focal segmental glomerulosclerosis and proteinuria associated with Myo1E mutations: novel variants and histological phenotype analysis.

BACKGROUND: Pathogenic mutations in the non-muscle single-headed myosin, myosin 1E (Myo1e), are a rare cause of pediatric focal segmental glomerulosclerosis (FSGS). These mutations are biallelic, to date only reported as homozygous variants in consanguineous families. Myo1e regulates the actin cytoskeleton dynamics and cell adhesion, which are especially important for podocyte functions. METHODS: DNA and RNA sequencing were used to identify novel MYO1E variants associated with FSGS. We studied the effects of these variants on the localization of Myo1e in kidney sections. We then analyzed the clinical and histological observations of all known pathogenic MYO1E variants. RESULTS: We identified a patient compound heterozygote for two novel variants in MYO1E and a patient homozygous for a deletion of exon 19. Computer modeling predicted these variants to be disruptive. In both patients, Myo1e was mislocalized. As a rule, pathogenic MYO1E variants map to the Myo1e motor and neck domain and are most often associated with steroid-resistant nephrotic syndrome in children 1-11 years of age, leading to kidney failure in 4-10 years in a subset of patients. The ultrastructural features are the podocyte damage and striking diffuse and global Alport-like glomerular basement membrane (GBM) abnormalities. CONCLUSIONS: We hypothesize that MYO1E mutations lead to disruption of the function of podocyte contractile actin cables resulting in abnormalities of the podocytes and the GBM and dysfunction of the glomerular filtration barrier. The characteristic clinicopathological data can help to tentatively differentiate this condition from other genetic podocytopathies and Alport syndrome until genetic testing is done. A higher resolution version of the Graphical abstract is available as Supplementary information.

The fusion oncogene VAV1-MYO1F triggers aberrant T-cell receptor signaling in vivo and drives peripheral T-cell lymphoma in mice.

VAV1-MYO1F is a recently identified gain-of-function fusion protein of the proto-oncogene Vav guanine nucleotide exchange factor 1 (VAV1) that is recurrently detected in T-cell non-Hodgkin's lymphoma (T-NHL) patients. However, the pathophysiological functions of VAV1-MYO1F in lymphomagenesis are insufficiently defined. Therefore, we generated transgenic mouse models to conditionally express VAV1-MYO1F in T-cells in vivo. We demonstrate that VAV1-MYO1F triggers cell autonomous activation of T-cell signaling with an activation of the ERK, JNK, and AKT pathways. VAV1-MYO1F expression induces a T-cell activation phenotype with high surface expression of CD25, ICOS, CD44, PD-1, and decreased CD62L as well as aberrant T-cell differentiation, proliferation, and neoplastic transformation. Consequently, the VAV1-MYO1F expressing T-cells induce a malignant T lymphoproliferative disease with 100% penetrance in vivo that mimics key aspects of human peripheral T-cell lymphoma. These results demonstrate that the human T-cell oncogene VAV1-MYO1F is sufficient to trigger oncogenic T-cell signaling and neoplastic transformation, and moreover, it provides a new clinically relevant mouse model to explore the pathogenesis of and treatment concepts for human T-cell lymphoma.

Myo1b promotes tumor progression and angiogenesis by inhibiting autophagic degradation of HIF-1α in colorectal cancer.

Myosin 1b (Myo1b) is an important single-headed membrane-associated motor of class I myosins that participate in many critical physiological and pathological processes. Mounting evidence suggests that the dysregulation of Myo1b expression has been extensively investigated in the development and progression of several tumors. However, the functional mechanism of Myo1b in CRC angiogenesis and autophagy progression remains unclear. Herein, we found that the expression of Myo1b was upregulated in CRC tissues and its high expression was correlated with worse survival. The overexpression of Myo1b promoted the proliferation, migration and invasion of CRC cells. Conversely, silencing of Myo1b suppressed tumor progression both in vitro and in vivo. Further studies indicated that Myo1b inhibited the autophagosome-lysosome fusion and potentiated the VEGF secretion of CRC cells to promote angiogenesis. Mechanistically, Myo1b blocked the autophagic degradation of HIF-1α and then led to the accumulation of HIF-1α, thus enhancing VEGF secretion and then promoting tumor angiogenesis in CRC. Together, our study provided novel insights into the role of Myo1b in CRC progression and revealed that it might be a feasible predictive biomarker and promising therapeutic target for CRC patients.

Steroid-Resistant Nephrotic Syndrome-Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.