Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning

BACKGROUND: During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). RESULTS: We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs. CONCLUSIONS: We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors.     

Comparative analysis of cis-regulation following stroke and seizures in subspaces of conserved eigensystems

Background  

It is often desirable to separate effects of different regulators on gene expression, or to identify effects of the same regulator across several systems. Here, we focus on the rat brain following stroke or seizures, and demonstrate how the two tasks can be approached simultaneously.     

Bio-mediated soil improvement

New, exciting opportunities for utilizing biological processes to modify the engineering properties of the subsurface (e.g. strength, stiffness, permeability) have recently emerged. Enabled by interdisciplinary research at the confluence of microbiology, geochemistry, and civil engineering, this new field has the potential to meet society's ever-expanding needs for innovative treatment processes that improve soil supporting new and existing infrastructure. This paper first presents an overview of bio-mediated improvement systems, identifying the primary components and interplay between different disciplines. Geometric compatibility between soil and microbes that restricts the utility of different systems is identified. Focus is then narrowed to a specific system, namely bio-mediated calcite precipitation of sands. Following an overview of the process, alternative biological processes for inducing calcite precipitation are identified and various microscopy techniques are used to assess how the pore space volume is altered by calcite precipitation, the calcite precipitation is distributed spatially within the pore space, and the precipitated calcite degrades during loading. Non-destructive geophysical process monitoring techniques are described and their utility explored. Next, the extent to which various soil engineering properties is identified through experimental examples. Potential advantages and envisioned applications of bio-mediated soil improvement are identified. Finally, the primary challenges that lie ahead, namely optimization and upscaling of the processes and the education/training of researchers/practitioners are briefly discussed.     

BNFinder: exact and efficient method for learning Bayesian networks

MOTIVATION: Bayesian methods are widely used in many different areas of research. Recently, it has become a very popular tool for biological network reconstruction, due to its ability to handle noisy data. Even though there are many software packages allowing for Bayesian network reconstruction, only few of them are freely available to researchers. Moreover, they usually require at least basic programming abilities, which restricts their potential user base. Our goal was to provide software which would be freely available, efficient and usable to non-programmers. RESULTS: We present a BNFinder software, which allows for Bayesian network reconstruction from experimental data. It supports dynamic Bayesian networks and, if the variables are partially ordered, also static Bayesian networks. The main advantage of BNFinder is the use exact algorithm, which is at the same time very efficient (polynomial with respect to the number of observations).     

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.Subject terms: Musculoskeletal abnormalities, Energy metabolism     

2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474

Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins. The complex peptide mixtures obtained were separated by 2-dimensional liquid chromatography coupled online with a nano-electrospray ionization ion trap mass spectrometer (2D LC/nanoESI-MS). The strong cation exchange column used in the first dimension of the separation was eluted in an automated fashion using a series of salt steps of increasing concentration. Peptides eluted from each of the salt steps were separated using a capillary reversed-phase HPLC column, the output of which was directed through a nano-electrospray fused silica tip into the mass spectrometer. Peptides were fragmented by collision-induced dissociation (CID) and analyzed by data-dependent MS/MS followed by database searching using the Sequest algorithm. Analysis of the data revealed both similarities and expected differences between proteins identified from these cell lines. As demonstrated by others, mRNA and the HER2/neu protein tyrosine kinase-linked receptor in BT474 cells is up regulated compared to its level in MCF7, while the expression of the estrogen receptor alpha is known to be up regulated in MCF7 cells. As expected, our studies showed identification of peptides from HER2 in BT474 while estrogen receptor peptides were detected in the MCF7 line. A total of 604 proteins were identified from BT474 membranes while 313 proteins were found from MCF7. The results are discussed in terms of the known differences in both protein and mRNA expression between these two breast cancer cell lines and also in the context of other known phenotypic differences between these cells.     

New insights into cytosolic glucose levels during differentiation of 3T3-L1 fibroblasts into adipocytes

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600μ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.