Maintenance Of Shape Research Articles

Evaluation of changes arising in the pig mesenchymal stromal cells transcriptome following cryopreservation and Trichostatin A treatment.

Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.

Maintenance of the anatomic contours in auricular reconstruction: The button technique

Background and Objective:Reconstructing the contours of the auricle is a unique challenge. Various bolster techniques have been tried to help prevent complications such as hematoma, seroma, and morbidity. Here, we describe a simple technique using a button to maintain the natural ear contour when it is at risk of a poor aesthetic outcome.Materials and Methods:A 77-year-old man underwent resection of a squamous cell carcinoma of the postauricular skin on the right ear, which involved the helical margin. A skin graft was chosen to close the defect. However, on initial inspection of the repair, buckling of the scaphoid fossa, collapse of the antihelical fold, and notching of the helix were observed. When these buckling changes persisted even after the anesthesia-related swelling resolved the following day, a button bolster was placed for 2.5 weeks to provide support for the cartilage.Results:Standardized digital imaging revealed maintenance of the original contours and sulci of the ear with an excellent cosmetic result.Conclusion:Recreation of the auricular contours is critical for an excellent cosmetic outcome. Using a button bolster is worth considering as it is of low cost, can easily fit into the natural ear contours, and can provide a rigid structure to ensure maintenance of the ear shape.

Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities

Basal-like breast cancer is an aggressive form of breast cancer with limited treatment options. The subgroup can be identified immunohistochemically, by lack of hormone receptor expression combined with expression of basal markers such as CK5/6 and/or epidermal growth factor receptor (EGFR). In vitro, several regulators of the actin cytoskeleton are essential for efficient invasion of basal-like breast cancer cell lines. Whether these proteins are expressed in vivo determines the applicability of these findings in clinical settings. The actin-regulating formin protein FHOD1 participates in invasion of the triple-negative breast cancer cell line MDA-MB-231. Here, we measure the expression of FHOD1 protein in clinical triple-negative breast cancers by using immunohistochemistry and further characterize the expression of another formin protein, INF2. We report that basal-like breast cancers frequently overexpress formin proteins FHOD1 and INF2. In cell studies using basal-like breast cancer cell lines, we show that knockdown of FHOD1 or INF2 interferes with very similar processes: maintenance of cell shape, migration, invasion, and proliferation. Inhibition of EGFR, PI3K, or mitogen-activated protein kinase activity does not alter the expression of FHOD1 and INF2 in these cell lines. We conclude that the experimental studies on these formins have implications in the clinical behavior of basal-like breast cancer.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

An ANN supported mathematical morphology based algorithm for lakes extraction from satellite images

With advances in remote sensing (RS) technology and platforms, more and more high-quality and fine spatial resolution satellite images are available. Manual method of feature extraction from remote sensing imagery is a tedious and time-consuming process. Thus automated and replicable technique plays vital role in updating lake database to evaluate the spatial and temporal evolution of lakes and ponds especially for vastly growing urban areas. This research work presents an artificial neural network (ANN) computed threshold value-based mathematical morphology (MM)-driven approach for extraction of lakes from satellite imageries with better accuracy. Accuracy of developed methodology has been assessed with the ground truths of the study area revealing better performance with different data-sets compared to existing methods. On an average scale for all data-sets used, the proposed algorithm is able to extract lakes with 99.47% accuracy and 0.9397 correlation coefficient (MCC). The existing classification method exhibited an accuracy of 98.75% and correlation coefficient of 0.89049. Similarly, the existing threshold-driven method has 99.31% accuracy and 0.90374 correlation coefficient. Maintenance of actual size and shape of the lakes, run-time control over structuring elements, semi-automation, faster processing, and single band adaptability are features of this work.

High protein and mRNA expression levels of TUBB3 (class III ß-tubulin) are associated with aggressive tumor features in esophageal adenocarcinomas.

BackgroundEsophageal adenocarcinomas show an increasing incidence in the Western world and their overall survival remains low. Microtubules are multifunctional cytoskeletal proteins involved in crucial cellular roles, including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Microtubulus-TUBB3 was found overexpressed in several carcinomas suggesting a significant role in cancer development. High levels of TUBB3 expression were also described to be associated with poor clinical outcome in various cancers. It was shown that overexpression of TUBB3 could be related to reduced efficiency of taxane-based targeting anticancer drugs in several cancer types.ResultsThere is a statistically significant association between high TUBB3 protein and TUBB3 mRNA expression and shortened survival (p<0,0001). Prognostic impact of TUBB3 expression is seen in patients with and without multimodal treatment. Multivariate analysis revealed a strong TUBB3 expression to be an independent prognosis factor. Validation of protein expression by mRNA in situ hybridization underlines the credibility of the immunohistochemical results.DiscussionOur study emphasized the significant importance of TUBB3 in esophageal adenocarcinoma. TUBB3 serves as an independent prognostic marker and may be a valuable biomarker for routine application in esophageal adenocarcinoma especially to address the need for adjuvant treatment in individuals following neoadjuvant therapy and surgery. Future prospective studies are needed which include the results of TUBB3 in preoperative biopsy material to proof the prognostic impact of TUBB3.Materials and Methods280 esophageal adenocarcinomas that underwent primary surgical resection or resection after neoadjuvant therapy were analyzed by mRNA-in-situ-hybridization (RNAscope®) and by immunohistochemistry (TUBB3 rabbit monoclonal antibody; Epitomics).

Taking control: reorganization of the host cytoskeleton by Chlamydia.

Both actin and microtubules are major cytoskeletal elements in eukaryotic cells that participate in many cellular processes, including cell division and motility, vesicle and organelle movement, and the maintenance of cell shape. Inside its host cell, the human pathogen Chlamydia trachomatis manipulates the cytoskeleton to promote its survival and enhance its pathogenicity. In particular, Chlamydia induces the drastic rearrangement of both actin and microtubules, which is vital for its entry, inclusion structure and development, and host cell exit. As significant progress in Chlamydia genetics has greatly enhanced our understanding of how this pathogen co-opts the host cytoskeleton, we will discuss the machinery used by Chlamydia to coordinate the reorganization of actin and microtubules.

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element.

The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other.IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.

Molecular architecture of the PBP2\u2013MreC core bacterial cell wall synthesis complex

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the “divisome”) and lateral wall growth (the “elongasome”). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.

Prevalence of βIII-tubulin (TUBB3) expression in human normal tissues and cancers.

Microtubules are multifunctional cytoskeletal proteins that are involved in crucial cellular roles including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Class III beta-tubulin (βIII-tubulin, also known as TUBB3) is a microtubule protein, normally expressed in cells of neuronal origin. Its expression was also reported in various other tumor types, such as several types of lung cancer, ovarian cancer, and esophageal cancer. TUBB3 is of clinical relevance as overexpression has been linked to poor response to microtubule-targeting anti-cancer drugs such as taxanes. To systematically investigate the epidemiology of TUBB3 expression in normal and neoplastic tissues, we used tissue microarrays for analyzing the immunohistochemically detectable expression of TUBB3 in 3911 tissue samples from 100 different tumor categories and 76 different normal tissue types. At least 1 tumor with weak expression could be found in 93 of 100 (93%) different tumor types, and all these 93 entities also had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in neurons of the brain, endothelium of blood vessels, fibroblasts, spermatogenic cells, stroma cells, endocrine cells, and acidophilic cells of the pituitary gland. In tumors, strong TUBB3 expression was most frequently found in various brain tumors (85%-100%), lung cancer (35%-80%), pancreatic adenocarcinoma (50%), renal cell carcinoma (15%-80%), and malignant melanoma (77%). In summary, these results identify a broad spectrum of cancers that can at least sporadically express TUBB3. Testing of TUBB3 in cancer types eligible for taxane-based therapies could be helpful to identify patients who might best benefit from this treatment.

High-resolution imaging of muscle attachment structures in Caenorhabditis elegans.

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in Caenorhabditis elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane [UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin), and UNC-95], are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed "zipper-like" structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the "zipper-like" structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have "dot-like" dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

In Situ Atomic Force Microscopic Studies of Single Tin Nanoparticle: Sodiation and Desodiation in Liquid Electrolyte.

Probing electrodes at a nanometer scale is challenging but desirable to reveal the structural evolution of materials in electrochemical reactions. Herein, we present an atomic force microscopic method for an in situ analysis of a single Sn nanoparticle during sodiation and desodiation, which is conducted in an aprotic liquid electrolyte akin to a real electrochemical environment of the Na-ion cells. The morphological evolution of different-sized single Sn nanoparticle is visualized during the charge/discharge cycles by using a homemade planar electrode. All of the Sn nanoparticles exhibit a dramatic initial volume expansion of about 420% after sodiation to Na15Sn4. Interestingly, we find that the smaller Sn nanoparticles show a lower rate of irreversible volume change and a better shape maintenance than the larger ones after desodiation. This finding suggests the importance of downsizing in improving the mechanical stability and the cycling performance of the Sn-based anodes in sodium-ion batteries.

Auricular Tissue Engineering Using Osteogenic Differentiation of Adipose Stem Cells with Small Intestine Submucosa.

Ear reconstruction remains a challenge for plastic surgeons. A tissue-engineering approach could provide another route for obtaining shape maintenance in neoauricular tissue. The authors designed a novel tissue-engineering auricular construct by culturing human adipose stem cells, which differentiated into osteocytes but not chondrocytes, in small intestine submucosa scaffolds. The authors evaluated cell growth potential and mechanical properties. An ear-shaped construct was created in vitro and then implanted in the backs of nude mice. The histology, cellularity, neovascularization, mechanical properties, and ear shape maintenance were investigated. In vitro, human adipose stem cells could be successfully seeded in the small intestine submucosa and differentiated toward osteogenesis. The ear-shaped human adipose stem cell/small intestine submucosa construct could maintain its shape in vivo up to 1 year. Alizarin Red S staining confirmed osteogenic differentiation. CD31 stain showed prominent angiogenesis in the human adipose stem cell/small intestine submucosa construct at 6 months and persistence up to 1 year. h-MHC stain revealed the maintenance of cellularity at 6 months and persistence up to 1 year. The mechanical properties were similar to those of native ear cartilage. The authors' study found that the combination of human adipose stem cells and small intestine submucosa could provide a more durable ear-shaped construct in vivo. The mechanical properties, shape, and cellularity were maintained in the constructs for up to 12 months. Therapeutic, V.

High-level βIII-tubulin overexpression occurs in most head and neck cancers but is unrelated to clinical outcome.

βIII-tubulin (TUBB3) is an isotype of microtubules, which are involved in crucial cellular roles including maintenance of cell shape, intracellular transport, and mitosis. Overexpression of TUBB3 was found to be associated with poor prognosis and resistance to tubulin-binding drugs and in several solid tumors including head and neck squamous cell carcinomas (HNSCC). Considering the potential high importance of a prognostic biomarker in these cancers, this study aimed to investigate the clinical relevance of immunohistochemical TUBB3 expression in HNSCC. Tissue microarray (TMA) sections containing samples from 667 cancers of oral cavity, oro- and hypopharynx, and larynx for which follow-up data were available were analyzed for TUBB3 expression by immunohistochemistry. Over 90% of our analyzed cancers showed unequivocal cytoplasmic TUBB3 expression. Staining was considered weak in 69 (15.5%), moderate in 149 (33.5%), and strong in 188 (42.2%) of cancers. The frequent TUBB3 overexpression showed no significant correlation with pathological grading, tumor stage, nodal status, or surgical margin and had no impact on patient outcomes. Despite lacking prognostic utility in HNSCC, the remarkable high prevalence of TUBB3 expression in HNSCC emphasizes its putative relevance as a target for future drugs targeting TUBB3.

Abstract 5114: Design, synthesis, and biological evaluation of tricyclic thieno[2,3- d]pyrimidines as microtubule targeting antitumor agents

Abstract Microtubules are dynamic structures that, together with actin microfilaments and intermediate filaments, constitute the cellular cytoskeleton. Besides their well-known roles in cell division, their functions involve maintenance of cell shape and morphology, cellular motility, and trafficking of organelles and vesicles. Recently, we reported a compound AG 370 as a microtubule targeting agent, that circumvented the Pgp and βIII-tubulin mediated drug resistance mechanisms that limit the efficacy of paclitaxel, docetaxel, and the vinca alkaloids. Molecular modeling and docking studies of the parent compound (AG 370) in the colchicine binding site (PDB: 402B) suggest that the C-5 and C-6 of parent compound are oriented towards the hydrophobic pockets with the side chain of Ala316, Val315, Leu255 and Met259. However, this binding pocket in tubulin is relatively large. To further explore the hydrophobic pocket, an additional cyclohexene ring was introduced at C-5 and C-6 of AG370. In the molecular modeling study, the resulting tricyclic scaffold showed hydrophobic interactions with the amino acids of the colchicine binding site. The 2-Me substituent of the tricyclic scaffold was replaced with a 2-H and a 2-NH2 group and 4-position of the scaffold was replaced with appropriate anilines. The 2-amino- N4-methoxyphenyl moiety (AG61) was found to be the most potent analog in the tricyclic series in both antiproliferative assay in human melanoma cancer cell line (MDA-MB-435 cell line, IC50 = 9.0 ± 0.2 nM) and in microtubule depolymerization assay in A-10 cells (EC50= 19 nM). Thus, we identified tricyclic thieno[2,3-d]pyrimidines as a novel structural scaffold with potent antiproliferative activity as well as microtubule depolymerizing activity. These analogs are selected for preclinical development. Citation Format: Farhana Islam, Aleem Gangjee, Xin Zhang, Xilin Zhou, Susan L. Mooberry. Design, synthesis, and biological evaluation of tricyclic thieno[2,3- d]pyrimidines as microtubule targeting antitumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5114. doi:10.1158/1538-7445.AM2017-5114

Photodynamic therapy with TMPyP – Porphyrine induces mitotic catastrophe and microtubule disorganization in HeLa and G361 cells, a comprehensive view of the action of the photosensitizer

Photodynamic therapy (PDT) is a useful tool against cancer and various other diseases. PDT is capable to induce different cell death mechanisms, due to the PDT evoked reactive oxygen species (ROS) production and is dose dependent. It is known that cytoskeleton is responsible for numerous cell functions, including cell division, maintenance of cell shape, their adhesion ability and movement. PDT initiated redistribution and subsequent disintegration of cytoskeletal components that precedes cell death. Here was present our results in HeLa and G361 cells subjected to sublethal PDT treatments using α,β,χ,δ porphyrin-Tetrakis (1-methylpyridinium-4-yl) p-Toluenesulfonate porphyrin (TMPyP). The photosensitizer (PS) induced transient increasing of mitotic index (MI) observable early after PDT, cell cycle arrest, microtubule (MTs) disorganization of interphase cells, aberrant mitosis and formation of rounded cells with partial loss of adherence. Some cells were partly resistant to PDT induced MTs disorganization. The differences between both cell lines to PDT response were described. This is the first evidence of TMPyP - PDT induced microtubule disorganization and the cell death mechanisms known as mitotic catastrophe and the first detail analysis of microtubule aberrations of mitotic and interphase cells in HeLa and G361 cell lines. New modification of techniques of protein immunolabeling was developed.

THE USE OF THE ORIGINAL METHOD OF RESTORING THE INFRAMAMMARY FOLD DURING TWO-STAGE BREAST RECONSTRUCTION

The Inframammary fold (IMF) is an important component of attractive and esthetically normal appearance of the breast and one of the most important elements of natural breast ptosis. It plays a significant role in the formation and maintenance of breast shape as well as it provides support for the implants placed subpectoral that prevent their malposition. It is necessary to restore the IMF during breast reconstruction and it allows achieving results similar to their natural breasts. The IMF reconstruction existing methods on a row with the advantages have some disadvantages: visible scars and contour irregularities in case of the external access, indentations at the site of IMF, little ptosis improvement or small projection, therefore it is impossible to choice a particular method of its reconstruction. Considering certain shortcomings of the existing methods of reconstruction Prof. A. Ismagilov et al. have developed a new IMF reconstruction technique using the posterior sheet of the expander capsule. This article presents our 4-year experience of performing this original technique during two-stage breast reconstruction.

Bacteria Getting into Shape: Genetic Determinants of E.coli Morphology.

ABSTRACTPerturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria. To understand the roles these genes play in defining morphology, 53 mutants with knockouts resulting in abnormal cell shape were crossed with the Keio collection in high throughput, generating 1,373 synthetic lethal interactions across 1.7 million double deletion mutants. This analysis yielded a highly populated interaction network spanning and linking multiple phenotypes, with a preponderance of interactions involved in transport, oxidation-reduction, and metabolic processes.

Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4.

The cell wall of free-living bacteria consists of peptidoglycan (PG) and is critical for maintenance of shape as dissolved solutes cause osmotic pressure and challenge cell integrity. Surprisingly, the subdivision 4 of the phylum Verrucomicrobia appears to be exceptional in this respect. Organisms of this subdivision are described to be devoid of muramic or diaminopimelic acid (DAP), usually found as components of PG in bacterial cell walls. Here we describe three novel bacterial strains from a freshwater lake, IG15T, IG16bT, and IG31T, belonging to a new genus in the subdivision 4 of Verrucomicrobia which we found to possess PG as part of their cell walls. Biochemical analysis revealed the presence of DAP not only in these novel strains, but also in Opitutus terrae PB90-1T, the closest described relative of strains IG15T, IG16bT, and IG31T. Furthermore, we found that nearly all genes necessary for peptidoglycan synthesis are present in genomes of subdivision 4 members, as well as in the complete genome sequence of strain IG16bT. In addition, we isolated and visualized PG-sacculi for strain IG16bT. Thus, our results challenge the concept of peptidoglycan-less free-living bacteria. Our polyphasic taxonomy approach places the novel strains in a new genus within the family Opitutaceae, for which the name Lacunisphaera gen. nov. is proposed. Strain designations for IG15T, IG16bT and IG31T are Lacunisphaera parvula sp. nov. (=DSM 26814 = LMG 29468), L. limnophila sp. nov. (=DSM 26815 = LMG 29469) and L. anatis sp. nov. (=DSM 103142 = LMG 29578) respectively, with L. limnophila IG16bT being the type species of the genus.

Targeting of Myosin to Cellular Organelles Results in Dramatic Reorganization based on Cargo/Motor Affinity

Unconventional myosins are vital molecular motors involved in active transport of cargo as well as maintenance of organelle shape. In particular, myosin VI has been shown to be involved in clathrin-mediated endocytosis and maintenance of golgi shape. Though the biophysical characteristics of unconventional myosins have been well studied and numerous cargo-binding partners have been identified, investigation into the interplay of these properties at the cellular level has been limited. Here, we investigate the influence of both monomeric and dimeric conformations of myosin VI, as well as the effects of binding affinity of the motor-cargo interaction may have on both subcellular morphology and cargo transport. We have generated a set of expressible fluorescent protein markers that label individual organelles/compartments. Using these markers combined with the FKBP-FRB system, we are able to selectively target myosin to peroxisomes (Pex3), early endosomes (Rab5), and clathrin-coated vesicles (Dab2) upon rapamycin stimulation. Irreversible localization of myosin to these structures results in disruption of normal compartment shape and size. Specifically, large clusters of peroxisomes and endosomes are observed and Dab2 labeled clathrin coated vesicles are disrupted and dispersed throughout the cell. In addition, these changes drastically remodel the cellular actin cytoskeletal network. These observations are seen using both artificially dimerized myosin VI and, to a lesser extent, monomeric myosin VI. Further, by controlling the strength of the FKBP-FRB interaction through mutagenesis, we have found that the strength of interaction between myosin and cargo plays a vital role in this reorganization.