The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.     

Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.     

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of the Performance of Information Theory-Based Methods and Cross-Correlation to Estimate the Functional Connectivity in Cortical Networks

Functional connectivity of in vitro neuronal networks was estimated by applying different statistical algorithms on data collected by Micro-Electrode Arrays (MEAs). First we tested these “connectivity methods” on neuronal network models at an increasing level of complexity and evaluated the performance in terms of ROC (Receiver Operating Characteristic) and PPC (Positive Precision Curve), a new defined complementary method specifically developed for functional links identification. Then, the algorithms better estimated the actual connectivity of the network models, were used to extract functional connectivity from cultured cortical networks coupled to MEAs. Among the proposed approaches, Transfer Entropy and Joint-Entropy showed the best results suggesting those methods as good candidates to extract functional links in actual neuronal networks from multi-site recordings.     

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1

We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.