Modelling cell motility and chemotaxis with evolving surface finite elements

We present a mathematical and a computational framework for the modelling of cell motility. The cell membrane is represented by an evolving surface, with the movement of the cell determined by the interaction of various forces that act normal to the surface. We consider external forces such as those that may arise owing to inhomogeneities in the medium and a pressure that constrains the enclosed volume, as well as internal forces that arise from the reaction of the cells' surface to stretching and bending. We also consider a protrusive force associated with a reaction-diffusion system (RDS) posed on the cell membrane, with cell polarization modelled by this surface RDS. The computational method is based on an evolving surface finite-element method. The general method can account for the large deformations that arise in cell motility and allows the simulation of cell migration in three dimensions. We illustrate applications of the proposed modelling framework and numerical method by reporting on numerical simulations of a model for eukaryotic chemotaxis and a model for the persistent movement of keratocytes in two and three space dimensions. Movies of the simulated cells can be obtained from http://homepages.warwick.ac.uk/maskae/CV_Warwick/Chemotaxis.html

Myosin VI Lever Arm Rotation: Fixed or Variable?

Two recent articles addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed1 or variable2. Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility

Age- and activity-related differences in the abundance of Myosin essential and regulatory light chains in human muscle

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM).We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patientderived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment

Occurrence and a possible mechanism of penetration of natural killer cells into k562 target cells during the cytotoxic interaction

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo)emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. \ud Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism. \u

Immunological Characterization of the Subunit of the 100 angstrom Filaments from Muscle Cells

We report the immunological characterization of the subunit of the intermediate sized (100 angstrom) filaments from muscle cells. The protein as isolated from smooth muscle (chicken gizzard) has an apparent molecular weight of 50,000. It is insoluble in buffers that solubilize myosin and the majority of actin, but becomes soluble in the presence of urea. Under a variety of experimental conditions, that include the presence of 8 M urea, this new protein comigrates with actin during purification studies. The two proteins can be separated from each other by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and antibodies have been elicited against the 50,000 dalton protein purified by using this technique. These antibodies crossreact with the partially purified protein in urea, but show no detectable cross reaction with actin or myosin. Indirect immunofluorescence reveals that in skeletal muscle this protein is found in close association with the Z lines of the sarcomeres and extends between the Z lines of adjacent myofibrils; it is also associated with filamentous structures that run along the length of a muscle fiber both in close association with the plasma membrane and between myofibrils. These filaments appear to connect myofibrils to each other or to the plasma membrane at the level of their Z lines. In heart muscle, the protein shows the same distribution as in skeletal muscle. In addition, it is found intimately associated with intercalated disks and areas of membrane interaction between laterally associated heart muscle cells. The immunofluorescent localization to the subunit of the 100 angstrom filaments suggests that in muscle cells this molecule may serve to link actin filaments at the level of the Z line (or intercalated disk) with the muscle plasma membrane. We believe that it functions in muscle primarily as a three dimensional matrix which interconnects individual myofibrils to one another and to the plasma membrane at the level of their Z lines. In this manner, this molecule may provide a framework that mechanically integrates all the contractile myofilaments during the contraction and relaxation of muscle. As a means of indicating its linking role in muscle, we have termed the protein desmin (from the Greek delta epsilon sigma µ os = link, bond)

Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei

BACKGROUND Vacuolar H-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. METHODS In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. RESULTS TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. CONCLUSIONS TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum

Methylated DNMT1 and E2F1 Are Targeted for Proteolysis by L3MBTL3 and CRL4DCAF5 Ubiquitin Ligase

Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4DCAF5. Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4DCAF5. Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated