Yingfan Zhang

Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A

We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease.

Nonmuscle myosin II exerts tension but does not translocate actin in vertebrate cytokinesis

During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.

Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice

ABSTRACT Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct–RFP and of Tpm3.1–mNeonGreen on secretory granules in LifeAct–RFP transgenic, Tpm3.1–mNeonGreen and myosin IIA–GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment. Summary: De novo assembly of functional actin–tropomyosin filaments in live rodents is investigated. Unlike non-muscle myosin II, tropomyosin is added to the actin filament shortly after nucleation.

Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells

Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.

Corrigendum: Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

Nature Communications 6: Article number: 7526 (2015); Published 25 June 2015; Updated 14 August 2015 Previous work by Kepiro et al. describing the photostable blebbistatin derivative para-nitroblebbistatin was inadvertently omitted from the reference list of this Article and should have been cited at instances where this inhibitor is referred to.

Emerging Role of Nonmuscle Myosin II and Implications for NMMII Associated Deafness

Objective: Data are scarce regarding the role of nonmuscle myosin II (NMMII) isoforms in the postnatal inner ear. Here we seek to: 1) understand the localization and function of each isoform in the postnatal environment, and 2) investigate the cause of hearing loss observed in mutations of NMMII. Method: Organ of Corti tissue was dissected from neonatal eGFP-NMMII-A or -B mice, and immunohistochemistry was performed using NMMII-isoform specific antibodies. OC cultures of rat were exposed to NMMII inhibitors at multiple time points. NMMII-C KO mice were stained with NMMII-isoform specific antibodies. ABRs on NMMII-A mutants were performed at various frequencies. Results: In WT mice, NMMII isoforms localize to the apical junction between hair and supporting cells. In eGFP-NMMII-A- or -B mice, NMMII isoform antibodies show a similar pattern of distribution, but are potentially expressed at different amounts. At the adhesion belt, the isoforms take on a “sarcomeric-like” pattern. NMMII inhibition results in variability of hair cell shape. In NMMII-C KO mice, NMMII-A demonstrates a new distribution and NMMII-B is upregulated. ABRs reveal an absence of hearing loss in NMMII-A D1424N and R702C mutant mice compared to controls. Conclusion: NMMII has a previously unrecognized “sarcomeric-like” pattern of organization, compatible with a contractile structure. Absence of hearing loss in NMMII-A mutants indicates that hearing loss may require a secondary insult, and isoforms have potential for compensation and overlap in function, which may also mitigate hearing loss.

Mouse Models of Human MYH9-Related Diseases

Point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain IIA (NMHC IIA), underlie autosomal dominant syndromes in humans (incidence 1 in 500,000). The abnormalities can manifest as macrothrombocytopenia, granulocyte inclusions, progressive proteinuric renal disease, cataracts, and sensorineural deafness. To gain insight into the pathological mechanism of MYH9-related diseases in humans, we generated mouse models of three disease-associated mutations, Arg702Cys in the amino-terminal domain of NMHC IIA which controls myosin motor activity, and Asp1424Gln and Glu1841Lys in the carboxyl-terminal rod domain, which regulates filament formation. Heterozygous Asp1424Gln and Glu1841Lys mutant mice produce homozygous mutant offspring at close to normal ratios. By contrast, homozygous Arg702Cys mice die at embryonic day E10.5 to E11.5 which though early, is considerably later in development than knockout NMHC IIA mice (E6.5). These results indicate that the motor domain function of NMHC IIA is critically important during the latter phase of mouse embryonic development. Giant platelets accompanied by decreased platelet counts and prolonged bleeding times are found in adult heterozygous and homozygous mice from all three mutant mouse lines. Bone marrow histology is consistent with failure of platelet release into the circulation. Some adult heterozygotes from all three mouse lines and homozygotes from Asp1424Gln and Glu1841Lys mouse lines have higher urine albumin/creatinine ratios than those of wild type controls. Light and transmission electron microscopy reveals focal segmental glomerulosclerosis with thicker basement membranes and abnormal podocytes. Some of the mutant mice also have lens abnormalities consistent with early cataract formation. Our results show that even heterozygous mutations in the mouse Myh9 gene can reproduce human MYH9-related diseases. These mouse models should be useful in understanding the pathophysiology of human MYH9-related diseases and also in designing and developing therapeutic stratagies.