CryoET reveals actin filaments within platelet microtubules.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

Lumenal components of cytoplasmic microtubules.

The lumen of cytoplasmic microtubules is a poorly explored expanse of intracellular space. Although typically represented in textbooks as a hollow tube, studies over several decades have shown that the microtubule lumen is occupied by a range of morphologically diverse components. These are predominantly globular particles of varying sizes which appear to exist either in isolation, bind to the microtubule wall, or form discontinuous columns that extend through the lumenal space. Actin filaments with morphologies distinct from the canonical cytoplasmic forms have also now been found within the microtubule lumen. In this review, we examine the historic literature that observed these lumenal components in tissues from diverse species and integrate it with recent cryo-electron tomography studies that have begun to identify lumenal proteins. We consider their cell and tissue distribution, possible mechanisms of incorporation, and potential functions. It is likely that continuing work in this area will open a new frontier in cytoskeletal biology.

In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Autoinhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects.

γ-Tubulin ring complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC-tethering proteins, including Drosophila Centrosomin (Cnn). Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here, we identify an extreme N-terminal CM1 autoinhibition (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits γ-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phosphomimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to γ-TuRCs is isoform specific and that misregulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is autoinhibited from binding γ-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to γ-TuRCs is regulated.

Impact of obesity on annual medical expenditures and diabetes care in Japanese patients with type 2 diabetes mellitus.

AIMS/INTRODUCTION: Diabetes and obesity are important health and economic concerns. We investigated the influence of obesity on diabetes control, the annual medical expenditures and medications in Japanese patients with type 2 diabetes who were relatively lean in comparison with those in Western countries. MATERIALS AND METHODS: A total of 402 Japanese patients with type 2 diabetes were enrolled and their annual medical expenditures investigated. Obesity was defined as body mass index ≥25 kg/m2 , according to the obesity classifications from the Japan Society for the Study of Obesity. RESULTS: A total of 165 patients (41.0%) were classified as obese. The obese group was younger, had poor glycemic control and higher frequency of hypertension than the non-obese group. The median total annual medical expenditures for all participants was ¥269,333 (interquartile range ¥169,664-437,437), which was equivalent to approximately $US2,450. The annual medical expenditure was significantly higher in patients with obesity than in non-obese patients (P < 0.001). This difference was mainly attributed to the annual expenditures for medication and hospitalization. In particular, the medication expenditures and the average number of drug classes for hyperglycemia and hypertension were significantly higher in the obese group. CONCLUSIONS: Japanese patients with type 2 diabetes and obesity had higher annual medical expenditures and a larger number of medications, but their diabetes control care was insufficient in comparison with those without obesity. Further studies are required to assess the effect of reducing bodyweight on diabetes control and costs.