Upon addition of GTPγS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I-derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme α1,2-mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10-fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPγS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf-1 as addition of a dominant, hydrolysis-deficient arf-1 Q 71 L mutant produced results similar to that of GTPγS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I-derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf-1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.

An increasing population of dementia patients produces substantial societal impacts. We assessed the prevalence of mild cognitive impairment (MCI) and all-cause dementia, including very mild dementia (VMD), in Taiwan. In a nationwide population-based cross-sectional survey, participants were selected by computerized random sampling from all 19 Taiwan counties and were enrolled between December 2011 and March 2013. Cases were identified through in-person interviews based on the National Institute on Aging-Alzheimer’s Association clinical criteria. Demographic data and histories involving mental status and function in daily living were collected. The principal objective assessments were the Taiwanese Mental Status Examination and Clinical Dementia Rating. In all, 10,432 people aged 65 years or older (mean age 76.2±6.7, 52.3% women) were interviewed. The age-adjusted prevalence of all-cause dementia was 8.04% (95% CI 7.47–8.61), including a 3.25% (95% CI 2.89–3.61) prevalence of VMD; that of MCI was 18.76% (95% CI 17.91–19.61). Women had a higher prevalence than men of both all-cause dementia (9.71% vs. 6.36%) and MCI (21.63% vs. 15.57%). MCI affects a considerable portion of the population aged 65 and above in Taiwan. The inclusion of VMD yields dementia prevalence rates higher than those previously reported from Taiwan. Old age, female gender, and a low educational level are significant associated factors.

Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.

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