Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372-384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.
When cells with a mesenchymal type of motility come into contact with an adhesive substrate they adhere and start spreading by the formation of lamellipodia. Using a label-free approach and virtual synchronization approach we analyzed spreading in fibroblasts and cancer cells. In all cell lines spreading is a non-linear process undergoing isotropic or anisotropic modes with first fast (5-20 min) and then slow (30-120 min) phases. In the first 10 min cell area increases 2-4 times, while the absolute rate of initial spreading decreases 2-8 times. Fast spreading depends on actin polymerization and dynamic microtubules. Inhibition of microtubule growth was sufficient for a slowdown of initial spreading. Inhibition of myosin II in the presence of stable microtubules restored fast spreading. Inhibition of actin polymerization or complete depolymerization of microtubules slowed down fast spreading. However, in these cases inhibition of myosin II only partially restored spreading kinetics. We conclude that rapid growth of microtubules towards cell margins at the first stage of cell spreading temporarily inhibits phosphorylation of myosin II and is essential for the fast isotropic spreading. Comparison of the fibroblasts with cancer cells shows that fast spreading in different cell types shares similar kinetics and mechanisms, and strongly depends on dynamic microtubules.
Paralogs CDK8 and CDK19 are regulatory kinases associated with the transcriptional Mediator complex. We have for the first time generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (DKO) males, but not single Cdk8 and Cdk19 KO, had an atrophic reproductive system and were infertile. The DKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star and Fads) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes likely associated with impaired synthesis of steroids. Star and Fads were also downregulated in cultivated Leydig cells after DKO. The treatment of primary Leydig cells culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as DKO, and prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. DKO, in contrast to single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CcnC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CcnC stabilization.
ABSTRACT During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, to reduce protein size, influencing the origin and evolution of NLSs and NoLSs in viruses.
В настоящее время для регенерации поврежденных тканей все шире используются мезенхимальные стволовые клетки.Способность к прикреплению и распластыванию на субстрате является фундаментальным свойством клеток, влияющим на процессы развития, органогенеза и гомеостаза организма.Распластывание клеток происходит, когда клетки прикрепляются к адгезивному субстрату за счет выдвижения тонкой ламеллиподии.Мы проанализировали процесс распластывания мезенхимальных стволовых клеток, используя виртуальную синхронизацию начала распластывания.Распластывание мезенхимальных стволовых клеток крупного рогатого скота (МСК КРС) -это нелинейный процесс, который включает в себя быструю фазу (0-10 минут) и медленную фазу распластывания (30 минут -3 часа).В первые десять минут клетки увеличивают свою площадь в 2-4 раза, а абсолютная начальная скорость распластывания уменьшается в 6,5 раз.Распластывание существенно замедляется при подавлении роста микротрубочек.В меньшей степени процесс распластывания замедляется при ингибировании полимеризации актина.При этом нарушение полимеризации микрофиламентов способствует изменению морфологии клеток
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