Characterization of Translocation Contact Sites Involved in the Import of Mitochondrial Proteins

Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner

Parametric Hidden Markov Models for Recognition and Synthesis of Movements

In humanoid robotics, the recognition and synthesis of parametric movements plays an extraordinary role for robot human interaction. Such a parametric movement is a movement of a particular type (semantic), for example, similar pointing movements performed at different table-top positions. For understanding the whole meaning of a movement of a human, the recognition of its type, likewise its parameterization are important. Only both together convey the whole meaning. Vice versa, for mimicry, the synthesis of movements for the motor control of a robot needs to be parameterized, e.g., by the relative position a grasping action is performed at. For both cases, synthesis and recognition, only parametric approaches are meaningful as it is not feasible to store, or acquire all possible trajectories. In this paper, we use hidden Markov models (HMMs) extended in an exemplar-based parametric way (PHMM) to represent parametric movements. As HMMs are generative, they are well suited for synthesis as well as for recognition. Synthesis and recognition are carried out through interpolation of exemplar movements to generalize over the parameterization of a movement class. In the evaluation of the approach we concentrate on a systematical validation for two parametric movements, grasping and pointing. Even though the movements are very similar in appearance our approach is able to distinguish the two movement types reasonable well. In further experiments, we show the applicability for online recognition based on very noisy 3D tracking data. The use of a parametric representation of movements is shown in a robot demo, where a robot removes objects from a table as demonstrated by an advisor. The synthesis for motor control is performed for arbitrary table-top positions

Translocation arrest by reversible folding of a precursor protein imported into mitochondria

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number

Fluorescence imaging for the anterior segment of the eye

Diagnostic technologies for the anterior segment of the eye, especially for hard-to-diagnose diseases such as microbial keratitis, are still lacking. Although in vivo confocal microscopy and optical coherence tomography are becoming more widely applicable to a variety of conditions, they are often prohibitively expensive, require specialized training and equipment, and are intrinsically insensitive to chemical changes. Here, ultraviolet-fluorescence imaging is proposed as a new technique to aid in investigation of the anterior segment. In this work, a novel two-color line-of-sight fluorescence imaging technique is described for imaging of the anterior segment. The technique is applied to seven ex vivo porcine eyes to illustrate the utility of the technique. The image data was used to estimate an effective fluorescence quantum yield of each eye at 370 nm. The eyes were then inoculated with bacteria to simulate microbial keratitis, a common sight-threatening infection, and the measurement was repeated. A simplified fluorescence-extinction model was developed to describe and analyze the relative intensities of the eye and biofilm fluorescence. Overall, the technique appears to have utility in clinical practice and with proper development may be suitable for detecting chemical changes in the eye, or the presence of foreign matter; however, further investigation is needed to develop the technique and analysis procedures into a quantitative diagnostic tool