Conversion of the Monomeric Red Fluorescent Protein into a Photoactivatable Probe

SummaryPhotoactivatable fluorescent proteins bring new dimension to the analysis of protein dynamics in the cell. Protein tagged with a photoactivatable label can be visualized and tracked in a spatially and temporally defined manner. Here, we describe a basic rational design strategy to develop monomeric photoactivatable proteins using site-specific mutagenesis of common monomeric red-shifted fluorescent proteins. This strategy was applied to mRFP1, which was converted into probes that are photoactivated by either green or violet light. The latter photoactivatable variants, named PA-mRFP1s, exhibited a 70-fold increase of fluorescence intensity resulting from the photoconversion of a violet-light-absorbing precursor. Detailed characterization of PA-mRFP1s was performed with the purified proteins and the proteins expressed in mammalian cells where the photoactivatable properties were preserved. PA-mRFP1s were used as protein tags to study the intracellular dynamics of GTPase Rab5

Lysosomal Targeting of Epidermal Growth Factor Receptors via a Kinase-dependent Pathway Is Mediated by the Receptor Carboxyl-terminal Residues 1022-1123

Binding of epidermal growth factor (EGF) to its receptor induces rapid internalization and degradation of both ligand and receptor via the lysosomal pathway. To study the mechanism of intracellular sorting of EGF-EGF receptor complexes to lysosomes, NIH 3T3 cells transfected with wild-type and mutant EGF receptors were employed. The kinetics of 125I-EGF trafficking was analyzed using low concentrations of the ligand to avoid saturation of the specific sorting system. The relative size of the pool of internalized 125I-EGF-receptor complexes that were capable of recycling decreased as receptors traversed the endosomal system. The rate of 125I-EGF sequestration from the recycling pathway correlated with the rate of 125I-EGF transition from early to late endosomes as measured by Percoll gradient fractionation. Deletion of the last 63 amino acids of the EGF receptor cytoplasmic tail did not inhibit the process of sequestration and targeting to the late endosomes and lysosomes. Truncation of the 123 residues, however, resulted in impaired lysosomal targeting and increased recycling of EGF. Receptor mutant in which 165 residues were deleted displayed maximal ability to recycle and a minimal extent of sorting to the late endosomes. The data suggest that two regions of the EGF receptor molecule, residues 1022-1063 and to a lesser extent residues 1063-1123, contribute in the regulation of routing of EGF receptors to the degradation pathway. The kinase-negative receptor mutant recycled EGF more intensively compared with the wild-type receptor, and the transport of this mutant to late endosomes was inhibited. These results support the view that the receptor kinase activity is important for ligand-induced sorting of EGF receptors to the pathway of lysosomal degradation

Development of molecular tools for expression and trafficking studies of the human monocarboxylate transporters

Most cancer cells rely on glycolysis to sustain their high proliferation rates with the production of lactate. For many years, lactate was seen as a metabolic waste of glycolytic metabolism in the tumor microenvironment, however, lactate has been recently associated as a key metabolic fuel and as an important signaling molecule 1,2. This substrate is responsible for extracellular acidification, which, is a feature of the tumor environment, and favors tumor invasion. The transport of lactate across the plasma membrane is mediated by a family of proton coupled monocarboxylate transporters (MCTs), which comprises 14 members 3. MCT1 and MCT4 serve as metabolic links between cancer cells via lactate exchange within tumors. This form of metabolic symbiosis illustrates how the apparent waste product from hypoxic tumor cells may be exploited by oxidative tumor cells to sustain their energy production under nutrient deprived conditions 4. MCTs are not only gatekeepers of intercellular metabolic cooperation, but also important regulators of angiogenesis and tumor migration, invasion and metastasis 5 . However, the role of MCTs in tumors is far from being well understood and their potential as therapeutic targets is poorly explored. Given the relationships between MCT1 and MCT4 in cancer cells, they offer a unique opportunity for novel treatment strategies. In this work, a set of molecular tools was generated for the expression and trafficking analyses of MCT1 and MCT4. Plasmids were designed harboring MCT1 or MCT4 with GFP or mCherry at the C- or N- terminal following the classical DNA cloning method. These molecular tools will be essential to study the expression and localization of MCT1 and MCT4 and to study the conditions and mechanisms underlying the endocytic trafficking of both transporters to further elucidate the significance of MCTs expression in tumor cells

Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor

Four independent mechanisms for uptake of activated EGFR are identified by a combination of receptor mutagenesis and RNA interference approaches

Formation of a Black String in a Higher Dimensional Vacuum Gravitational Collapse

We present a solution to the vacuum Einstein Equations which represents a collapse of a gravitational wave in 5 dimensions. Depending on the focal length of the wave, the collapse results, either in a black string covered by a horizon, or in a naked singularity which can be removed.Comment: Minor changes. A reference added. Matches the print version. To appear in Phys. Lett.

Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein Kinase C-dependent modulation

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes

Dimension on Discrete Spaces

In this paper we develop some combinatorial models for continuous spaces. In this spirit we study the approximations of continuous spaces by graphs, molecular spaces and coordinate matrices. We define the dimension on a discrete space by means of axioms, and the axioms are based on an obvious geometrical background. This work presents some discrete models of n-dimensional Euclidean spaces, n-dimensional spheres, a torus and a projective plane. It explains how to construct new discrete spaces and describes in this connection several three-dimensional closed surfaces with some topological singularities It also analyzes the topology of (3+1)-spacetime. We are also discussing the question by R. Sorkin [19] about how to derive the system of simplicial complexes from a system of open covering of a topological space S.Comment: 16 pages, 8 figures, Latex. Figures are not included, available from the author upon request. Preprint SU-GP-93/1-1. To appear in "International Journal of Theoretical Physics

Endocytosis: A Turnover Mechanism Controlling Ion Channel Function

Ion channels (IChs) are transmembrane proteins that selectively drive ions across membranes. The function of IChs partially relies on their abundance and proper location in the cell, fine-tuned by the delicate balance between secretory, endocytic, and degradative pathways. The disruption of this balance is associated with several diseases, such as Liddle’s and long QT syndromes. Because of the vital role of these proteins in human health and disease, knowledge of ICh turnover is essential. Clathrin-dependent and -independent mechanisms have been the primary mechanisms identified with ICh endocytosis and degradation. Several molecular determinants recognized by the cellular internalization machinery have been discovered. Moreover, specific conditions can trigger the endocytosis of many IChs, such as the activation of certain receptors, hypokalemia, and some drugs. Ligand-dependent receptor activation primarily results in the posttranslational modification of IChs and the recruitment of important mediators, such as β-arrestins and ubiquitin ligases. However, endocytosis is not a final fate. Once internalized into endosomes, IChs are either sorted to lysosomes for degradation or recycled back to the plasma membrane. Rab proteins are crucial participants during these turnover steps. In this review, we describe the major ICh endocytic pathways, the signaling inputs triggering ICh internalization, and the key mediators of this essential cellular process