Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14

Interpolar microtubules are sorted by the directional instability resulting from antagonistic molecular motors, not a stable balance of force

Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets

Compartmentalization in a prebiotic setting is an important aspect of early cell formation and is crucial for the development of an artificial protocell system that effectively couples genotype and phenotype. Aqueous two-phase systems (ATPSs) and complex coacervates are phase separation phenomena that lead to the selective partitioning of biomolecules and have recently been proposed as membrane-free protocell models. We show in this study through fluorescence recovery after photobleaching (FRAP) microscopy that despite the ability of such systems to effectively concentrate RNA, there is a high rate of RNA exchange between phases in dextran/polyethylene glycol ATPS and ATP/poly-L-lysine coacervate droplets. In contrast to fatty acid vesicles, these systems would not allow effective segregation and consequent evolution of RNA, thus rendering these systems ineffective as model protocells. Electronic supplementary material The online version of this article (doi:10.1007/s11084-014-9355-8) contains supplementary material, which is available to authorized users

Poleward transport of Eg5 by dynein–dynactin in Xenopus laevis egg extract spindles

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization

Controlled Growth of Filamentous Fatty Acid Vesicles under Flow

The earliest forms of cellular life would have required a membrane compartment capable of growth and division. Fatty acid vesicles are an attractive model of protocell membranes, as they can grow into filamentous vesicles that readily divide while retaining their contents. In order to study vesicle growth, we have developed a method for immobilizing multilamellar fatty acid vesicles on modified glass surfaces and inducing filamentous membrane growth under flow. Filament formation strictly depended on the presence of freshly neutralized fatty acid micelles in the flow chamber. Using light microscopy, we observed a strong dependence of initial growth velocity on initial vesicle size, suggesting that new fatty acid molecules were incorporated into the membrane over the entire external surface of the vesicle. We examined the influences of flow rate, fatty acid concentration, and salt concentration on filamentous growth and observed drastic shape changes, including membrane pearling, of preexisting membrane tubules in response to osmotic stress. These results illustrate the versatility of flow studies for exploring the process of fatty acid vesicle growth following exposure to free fatty acids

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

BACKGROUND:Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS:We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE:These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein

Questioning the Value of Fluorodeoxyglucose Positron Emission Tomography for Residual Lesions After Chemotherapy for Metastatic Seminoma: Results of an International Global Germ Cell Cancer Group Registry

Purpose Residual lesions after chemotherapy are frequent in metastatic seminoma. Watchful waiting is recommended for lesions < 3 cm as well as for fluorodeoxyglucose (FDG) positron emission tomography (PET)-negative lesions ≥ 3 cm. Information on the optimal management of PET-positive residual lesions ≥ 3 cm is lacking. Patients and Methods We retrospectively identified 90 patients with metastatic seminoma with PET-positive residual lesions after chemotherapy. Patients with elevated α-fetoprotein or nonseminomatous histology were excluded. We analyzed the post-PET management and its impact on relapse and survival and calculated the positive predictive value (PPV) for PET. Results Median follow-up time was 29 months (interquartile range [IQR], 10 to 62 months). Median diameter of the largest residual mass was 4.9 cm (range, 1.1 to 14 cm), with masses located in the retroperitoneum (77%), pelvis (16%), mediastinum (17%), and/or lung (3%). Median time from the last day of chemotherapy to PET was 6.9 weeks (IQR, 4.4 to 9.9 weeks). Post-PET management included repeated imaging in 51 patients (57%), resection in 26 patients (29%), biopsy in nine patients (10%) and radiotherapy in four patients (4%). Histology of the resected specimen was necrosis in 21 patients (81%) and vital seminoma in five patients (19%). No biopsy revealed vital seminoma. Relapse or progression occurred in 15 patients (17%) after a median of 3.7 months (IQR, 2.5 to 4.9 months) and was found in 11 (22%) of 51 patients on repeated imaging, in two (8%) of 26 patients after resection, and in two (22%) of nine patients after biopsy. All but one patient who experienced relapse were successfully treated with salvage therapy. The PPV for FDG-PET was 23%. Conclusion FDG-PET has a low PPV for vital tumor in residual lesions after chemotherapy in patients with metastatic seminoma. This cautions against clinical decisions based on PET positivity alone

Outcomes of men with HIV and germ cell cancer : Results from an international collaborative study

Background: Previous studies have shown that men with HIV and germ cell cancer (HIV-GCC) have inferior overall survival (OS) in comparison with their HIV-negative counterparts. However, little information is available on treatments and outcomes of HIV-GCC in the era of combination antiretroviral therapy (cART). Methods: This study examined men living with HIV who were 18 years old or older and had a diagnosis of histologically proven germ cell cancer (GCC). The primary outcomes were OS and progression-free survival (PFS). Results: Data for 89 men with a total of 92 HIV-GCCs (2 synchronous GCCs and 1 metachronous bilateral GCC) were analyzed; among them were 64 seminomas (70%) and 28 nonseminomas (30%). The median age was 36 years, the median CD4 T-cell count at GCC diagnosis was 420 cells/µL, and 77% of the patients on cART had an HIV RNA load < 500 copies/mL. Stage I disease was found in 44 of 79 gonadal GCCs (56%). Among 45 cases with primary disseminated GCC, 78%, 18%, and 4% were assigned to the good-, intermediate-, and poor-prognosis groups, respectively, of the International Germ Cell Cancer Collaborative Group. Relapses occurred in 14 patients. Overall, 12 of 89 patients (13%) died. The causes of death were refractory GCC (n = 5), an AIDS-defining illness (n = 3), and other causes (n = 4). After a median follow-up of 6.5 years, the 5- and 10-year PFS rates were 81% and 73%, respectively, and the 5- and 10-year OS rates were 91% and 85%, respectively. Conclusions: The 5- and 10-year PFS and OS rates of men with HIV-GCC were similar to those reported for men with HIV-negative GCC. Patients with HIV-GCC should be managed identically to HIV-negative patients. Lay Summary: Men living with HIV are at increased risk for germ cell cancer (GCC). Previous studies have shown that the survival of men with HIV-associated germ cell cancer (HIV-GCC) is poorer than the survival of their HIV-negative counterparts. This study examined the characteristics, treatments, and outcomes of 89 men with HIV-GCC in the era of effective combination antiretroviral therapies. The long-term outcomes of men with HIV-GCC were similar to those reported for men with HIV-negative GCC. Patients with HIV-GCC should be managed identically to HIV-negative patients