Interaction between adaptor proteins Ruk/CIN85 and Tks4 in normal and tumor cells of different tissue origins

Aim. To study the interaction of adaptor protein Ruk/CIN85 SH3 domains with endogenous adaptor protein Tks4 in normal and tumor cells of different tissue origins. Methods. GST in vitro pull-down assay was performed using total cell lysates of cell lines of different origins. Results. Using GST in vitro pull-down assay, we have determined that SH3A domain of adaptor protein Ruk/CIN85 precipitated full-length form of adaptor protein Tks4 (Mr 120 kDa) from lysates of human breast (MCF-7, MDA-MB-231), melanoma (MM-4), colon (HT-29, DLD-1) tumor cells as well as from lysates of mouse Lewis lung carcinoma cells (LLC) and fibroblasts (NIH 3T3). It has been also revealed that all Ruk/CIN85 SH3 domains (A, B and C) with high efficiency precipitate the additional forms of Tks4 with Mr 75, 90 and 160 kDa from lysates of human colon carcinoma cells and mouse fibroblasts. The molecular nature of new multiple forms of Tks4 has not been determined to date. Conclusions. New multiple molecular forms of adaptor protein Tks4 with Mr 75, 90 and 160 kDa, able to interact with high affinity with SH3 domains of Ruk/CIN85, were identified using GST in vitro pull-down assay. The data obtained suggest that interaction between Ruk/CIN85 SH3 domains with Tks4 endogenous forms is determined by cellular context while a level of this interaction can be regulated in the course of physiological cellular responses.Мета. З’ясувати особливості взаємодії окремих доменів SH3 адаптерного білка Ruk/CIN85 з ендогенним адаптерним білком Tks4 у нормальних і пухлинних клітинах різного тканинного походження. Методи. Аналіз GST in vitro pull-down проводили з використанням загальних клітинних лізатів ліній клітин різного тканинного походження. Результати. Встановлено, що домен SH3А адаптерного білка Ruk/CIN85 преципітує повнорозмірну форму адаптерного білка Tks4 (Mr 120 кДа) з лізатів пухлинних клітин молочної залози (MCF-7, MDA-MB-231), меланоми (MM-4), ободової кишки (HT-29, DLD-1) людини, а також карциноми легені Льюїс (LLC) та фібробластів (NIH 3T3) миші. Виявлено також, що всі домени SH3 білка Ruk/CIN85 (А, В і С) з високою ефективністю преципітують додаткові форми Tks4 з Mr 75, 90 і 160 кДа з лізатів клітин карциноми ободової кишки людини та фібробластів миші. Молекулярну природу нових множинних форм Tks4 на сьогодні не встановлено. Висновки. Ідентифіковано нові множинні молекулярні форми адаптерного білка Tks4 з Mr 75, 90 і 160 кДа, здатні з високою афінністю взаємодіяти з доменами SH3 білка Ruk/CIN85. Отримані дані дозволяють припустити, що можливість зв’язування доменів SH3 Ruk/CIN85 з ендогенними формами білка Tks4 обумовлена клітинним контекстом, тоді як рівень взаємодії регулюється в процесі реалізації фізіологічних відповідей клітин.Цель. Выяснить особенности взаимодействия отдельных доменов SH3 адаптерного белка Ruk/CIN85 с эндогенным адаптерным белком Tks4 в нормальных и опухолевых клетках разного тканевого происхождения. Методы. Анализ GST in vitro pull-down проводили с использованием общих клеточных лизатов линий клеток разного тканевого происхождения. Результаты. Установлено, что домен SH3А адаптерного белка Ruk/CIN85 преципитирует полноразмерную форму адаптерного белка Tks4 (Mr 120 кДа) из лизатов опухолевых клеток молочной железы (MCF-7, MDAMB-231), меланомы (MM-4), ободочной кишки (HT-29, DLD-1) человека, а также карциномы легкого Льюис (LLC) и фибробластов (NIH 3T3) мыши. Выявлено также, что все домены SH3 белка Ruk/CIN85 (А, В и С) с высокой эффективностью преципитируют дополнительные формы Tks4 с Mr 75, 90 и 160 кДа из лизатов клеток карциномы ободочной кишки человека и фибробластов мыши. Молекулярная природа новых множественных форм Tks4 на сегодня не установлена. Выводы. При помощи анализа GST in vitro pull-down идентифицированы новые множественные молекулярные формы адаптерного белка Tks4 с Mr 75–90 и 160 кДа, способные с высокой аффинностью взаимодействовать с доменами SH3 белка Ruk/CIN85. Полученные данные позволяют предположить, что возможность взаимодействия доменов SH3 белка Ruk/CIN85 с эндогенными формами белка Tks4 определяется клеточным контекстом, в то время как уровень связывания регулируется в процессе реализации физиологических ответов клеток

A Mutation in Myo15 Leads to Usher-Like Symptoms in LEW/Ztm-ci2 Rats

The LEW/Ztm-ci2 rat is an animal model for syndromal deafness that arose from a spontaneous mutation. Homozygous animals show locomotor abnormalities like lateralized circling behavior. Additionally, an impaired vision can be observed in some animals through behavioral studies. Syndromal deafness as well as retinal degeneration are features of the Usher syndrome in humans. In the present study, the mutation was identified as a base substitution (T->C) in exon 56 of Myo15, leading to an amino acid exchange from leucine (Leu) to proline (Pro) within the carboxy-terminal MyTH4 domain in the proteins' tail region. Myo15 mRNA was expressed in the retina as demonstrated for the first time with the help of in-situ hybridization and PCR. To characterize the visual phenotype, rats were examined by scotopic and photopic electroretinography and, additionally, histological analyses of the retinas were conducted. The complete loss of sight was detected along with a severe degeneration of photoreceptor cells. Interestingly, the manifestation of the disease does not solely depend on the mutation, but also on environmental factors. Since the LEW/Ztm-ci2 rat features the entire range of symptoms of the human Usher syndrome we think that this strain is an appropriate model for this disease. Our findings display that mutations in binding domains of myosin XV do not only cause non-syndromic hearing loss but can also lead to syndromic disorders including retinal dysfunction

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release

A kinase anchoring protein 9 is a novel myosin VI binding partner that links myosin VI with the PKA pathway in myogenic cells

10.1155/2015/816019BioMed Research International201581601

Myosin VI in skeletal muscle: Its localization in the sarcoplasmic reticulum, neuromuscular junction and muscle nuclei

10.1007/s00418-012-1070-9Histochemistry and Cell Biology1396873-88

Myosin VI Localization and Expression in Striated Muscle Pathology

We study the solutions of the matrix equation $S\exp(S) = A$. Our motivation comes from the study of systems of delay differential equations $y'(t) = A y(t-1)$, which occur in some models of practical interest, especially in mathematical biology. This paper concentrates on the distinction between \emph{evaluating a matrix function} and \emph{solving a matrix equation}. In particular, it shows that the matrix Lambert $W$ function evaluated at the matrix $A$ does not represent all possible solutions of $S\exp(S) = A$. These results can easily be extended to more general matrix equations

BAG3-related myopathy, polyneuropathy and cardiomyopathy with long QT syndrome

10.1007/s10974-015-9431-3Journal of Muscle Research and Cell Motility366423-43