Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis.

The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.

Structure of the fission yeast actomyosin ring during constriction.

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.

Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast.

Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts.

Efficacy of nutritional supplement (Haras) on carbon monoxide levels in smokers and non-smokers: An observational study.

Context: Carbon monoxide (CO) concentrations in exhaled air may impart a quick, non-invasive method to determine smoking status. Haras is a nutraceutical medication, which is slowly gaining recognition for its antioxidant and anti-inflammatory activities. Aims: The effectiveness of the Haras therapy in smokers and non-smokers will be assessed by evaluating breath CO levels. Methods and Materials: The study included 101 test subjects with 76 subjects of smokers and 25 subjects of non-smokers. Both the test groups were given 10 mL of Haras juice in divided doses per day for 30 days. The CO levels were evaluated using a breath analyser before drug trial and then on the 8th, 15th, 22nd and after the conclusion of the drug trial. Statistical Analysis Used: The Wilcoxon signed-rank test was used to compare the CO and carboxyhemoglobin levels among smokers and non-smokers. Results: Smokers had higher mean percent carboxyhemoglobin and mean parts per million CO values than non-smokers, and the difference between the two was shown to be statistically significant (P < 0.001). It was also found to be statistically significant from the first day to the eighth day, the first day to the 15th day, the first day to the 20th second day, first day to the 30th day (P < 0.001). Conclusions: Haras can be used effectively as an alternative supportive treatment for the diminution of CO levels in smokers and non-smokers.

Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Ewing's sarcoma of the mandible with multilocular radiolucency.

Ewing's sarcoma (ES) is a small blue round cell tumor, malignant in nature typically affecting long bones and pelvis. Occurrence of ES in the head and neck region is rare and is reported to be 2%-3%, of which the chances of having primary lesion are rare. In the head and neck region, it has been reported to strike skull, supraclavicular region, parotid region, orbital floor, nasal cavity, maxilla, mandible and zygoma. We present a case of primary ES of the mandible in a 22-year-old female who reported with a palpable swelling on the lower left part of the face; and intraorally, there was a growth in the molar region. Orthopantomogram showed multilocular radiolucency in the molar-ramus region. The diagnosis was made after surgical resection, histopathology and immunohistochemistry profiling. The patient was treated by cortical segmental resection of mandible combined with chemotherapy and a follow-up was done for 2 years.

Cytokinesis: catch and drag.

Recent studies of actomyosin-ring assembly in fission yeast have suggested that an intricate web of membrane-bound nodes containing myosin and the actin nucleator formin is pulled together into a tight ring through a 'search-and-capture' mechanism.

Histopathologic and immunohistochemical findings of odontogenic jaw cysts treated by decompression technique.

CONTEXT: Odontogenic cysts are among the most common lesions to affect the oral and maxillofacial region. Cysts are capable of causing significant bony disfigurement, tooth displacement and pathological fractures. Several surgical approaches exist for the management of larger cysts of the jaws. These include enucleation, marsupialization and decompression. AIMS: 1. Analysis of histopathologic findings in odontogenic cysts before and after decompression2. Analysis of Ki-67 expression in odontogenic jaw cysts before and after decompression. SETTINGS AND DESIGN: Decompression technique was used for the treatment of 10 cases of odontogenic cysts in the study. Incisional biopsies of cystic lining (pretreatment) and corresponding excisional biopsies (posttreatment) were received for histopathologic and immunohistochemical examination. SUBJECTS AND METHODS: Hematoxylin and eosin stain was used for histopathologic findings, and Ki-67 was used for immunohistochemical findings using antibody Ki-67 in fresh tissue samples. RESULTS: Overall, radicular cysts, dentigerous cysts, and sialo-odontogenic cyst contained fewer Ki-67 + cells than odontogenic keratocysts. The average scores were found to be 2.2 and 1 for before and after decompression, respectively. A statistically significant difference was observed between the two groups. The two-tailed P value was found to be <0.0001. The confidence interval was found to be 95%. CONCLUSIONS: The proliferative activity evaluated by Ki-67 marker was greater in predecompression epithelial lining compared to postdecompression. Our study infers that proliferative rate of the cystic epithelial lining is significantly diminished after decompression.

Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

The Current Understanding on Langerhans' Cells and Its Role in Oral Lesions.

OBJECTIVE: Description of Langerhans' cells (LCs) as an important antigen-presenting cells responsible for detecting the antigens, recruiting T-cells, and thereby initiating the immune response. An adequate response of the mucosal immune system is essential to protect the mucosa against pathological conditions. Hence, a detailed review was planned about this unique antigen-presenting cell. METHODS: A literature search of the electronic databases included the MEDLINE, EBSCOHOST, PUBMED, and hand searches of references retrieved were undertaken using the following MeSH terms "Langerhans cells," "LCs in Oral Lichen Planus," "Langerhans cell histiocytosis," "LCs and HIV," "LCs in Periodontitis." RESULTS: LCs are present suprabasally in the epithelium of oral mucosa and in the epidermis of the skin. The role played by LCs though not fully elucidated, but several research studies indicate that these cells are involved in the pathogenesis of many oral diseases. In this article, the historical perspective, structure, function, origin, and phenotypic expressions of LCs are discussed in detail. The current understanding on the role of LCs in various oral lesions and its immunological characteristics are discussed. CONCLUSION: LCs act as immune mediator cells, tumor cells, vectors of infected cells, and phagocytic cells. Further studies could bolster the knowledge about the role of Langerhans cells in the immune response of various oral diseases and thereby provide diagnostic tools and help for prognostic evaluation. This review illuminates the pivotal role of Langerhans cells and its immune surveillance as a "Sentinels" of the oral mucosa.

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast.

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.

Structure, function and role of CD44 in neoplasia.

CD44 is a group of protein molecules which perform a variety of functions. Their wide range of functions are mainly based on their multiple variations in their molecular structure. Furthermore, they are distributed in various tissues of the human body. They have a unique property of cell adhesion, which can lead to interaction between two different cells or a cell and its pericellular matrix. CD44 as a cell surface adhesive molecule helps in aggregation and migration of tumor cells. CD44 plays an important role in cancer of bladder, liver, lungs, pancreas, etc. Expression profile of CD44 has been seen in the epithelia of the lip, tongue, gingiva, hard palate, floor of the mouth, buccal mucosa and pharynx. The relationship between the expression of CD44 v6 and regional lymph node metastasis has been studied immunohistochemically. The expression of CD44 v6 was apparently downregulated in oral squamous cell carcinoma, but not in normal oral mucosa. Carcinomas expressing lower levels of CD44 v6 exhibited more frequent regional lymph node metastasis. No significant relation was found between the expression of CD44 v6 in primary and metastatic lesions. Still, the precise function of CD44 in the metastatic process and the degree of involvement in human malignancies is yet to be established.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast.

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.

Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins.

Cytokinesis in many eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by the existing literature and new three-dimensional (3D) molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin cross-linkers, and membranes and simulate their interactions. Assuming that local force on the membrane results in inward growth of the cell wall, we explored a matrix of possible actomyosin configurations and found that node-based architectures like those presently described for ring assembly result in membrane puckers not seen in electron microscope images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses.

A rare case report of craniofacial fibrous dysplasia.

Fibrous dysplasia (FD) is a fibro-osseous lesion of the osseous structures of the body. The exact cause is unknown; however, recently, the cause has been reported to be postzygomatic somatic mutation in guanine nucleotide-binding protein, alpha stimulating 1 gene located at chromosome 20q13.2. The three subtypes of FD are monostotic, polyostotic and craniofacial. The term craniofacial FD (CFD) is used to describe FD where the lesions are confined to contiguous bones of the craniofacial skeleton. This report describes the case of CFD of a 20-year-old male patient who had unusual presentation involving right maxilla and frontal bone of the left side of the face. The clinical features, radiological findings and treatment have been discussed.

Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe.

Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.

Isolation of Cytokinetic Actomyosin Rings from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Cytokinesis is the final stage of cell division, through which cellular constituents of mother cells are partitioned into two daughter cells resulting in the increase in cell number. In animal and fungal cells cytokinesis is mediated by an actomyosin contractile ring, which is attached to the overlying cell membrane. Contraction of this ring after chromosome segregation physically severs the mother cell into two daughters. Here we describe methods for the isolation and partial purification of the actomyosin ring from the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which can serve as in vitro systems to facilitate biochemical and ultrastructural analysis of cytokinesis in these genetically tractable model systems.

Immunomorphological patterns of cervical lymph nodes in oral squamous cell carcinoma.

CONTEXT: Squamous cell carcinoma (SCC) comprises 80% of the cancers of the oral cavity. Metastases to the cervical lymph nodes affects prognosis. Studying lymph node reactivity may help to understand host immune reaction against the tumor and thus influence prognosis. AIMS: This study observed patterns of lymph node reactivity, metastases and grades of metastases in cervical lymph nodes and compared them with the histopathology of the primary tumor. MATERIALS AND METHODS: Lymph nodes from 30 patients of oral squamous cell carcinoma (OSCC) were taken. 10 of these were well-differentiated SCC (WDSCC), 10 moderately differentiated SCC (MDSCC) and 10 poorly differentiated SCC (PDSCC). Four immunomorphological patterns were observed: lymphocyte predominance, germinal centre predominance, mixed pattern (sinus histiocytosis) and unstimulated pattern. STATISTICAL ANALYSIS USED: Chi square test. RESULTS: The predominant lymph node reactive patterns were germinal centre predominance (79.27%), unstimulated pattern (14.63%) and lymphocyte predominance (6.10%). Positive nodes showed invasion in the form of islands (57.14%), cords (39.29%) and total replacement pattern (3.57%). Tumor involvement in positive nodes showed grade 3 invasion, (53.57%), grade 2 invasion (26.79%), grade 4 invasion (17.86%) and grade 1 invasion (1.79%). Statistically significant association was found between: Lymph node reactive pattern and histopathological grade of primary tumor. Higher numbers of germinal centre predominance lymph nodes were found in WDSCC and MDSCCHistopathological grade of primary tumor and the grade of lymph node invasion. CONCLUSIONS: Immuno-morphological assessment of draining lymph nodes reflects the immune status of the patient with respect to metastases. This may facilitate identification of high and low risk patients and help in planning appropriate therapy for the high-risk patients.

The yeast actin cytoskeleton.

The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.