Rab21CA over-expression is associated with increased invasion and matrix degradation.

<p><b>A.</b> Trophozoites stably expressing Rab21WT, Rab21KD, Rab21CA and Rab21DN and vector control (pEhExHA for Rab21WT and mutants; psAP2Gunma for Rab21KD strain) were harvested, washed and resuspended in serum free BI medium and 7.5 x10⁵ cells were plated in upper chamber of a transwell insert coated with 5mg/ml of matrigel. The cells were attracted in the lower chamber using 15% adult bovine serum as a chemoattractant. Trophozoites were allowed to invade for 48–50hrs.After 48hrs, the migrated cells in the lower chamber were detached and counted. Graph shows mean ± SEM of three independent experiments. <b>B.</b> Trophozoites stably expressing Rab21CA and Rab21DN were plated on glass coverslips coated with Hilyte488 Fibronectin and incubated at 35°C for 45–48 hrs under dark. After 48hrs, the cells were fixed and stained with Alexa568 Phalloidin for actin and confocal images were acquired using Zeiss LSM780. Scale bars, 10μm.</p

Rab21 regulates the amoebic actin dots.

<p><b>A.</b> Trophozoites stably expressing HA tagged Rab21WT, Rab21CA and Rab21DN were harvested and plated on uncoated glass surface, fixed and stained using anti HA, followed by secondary anti mouse Alexa488 and Alexa568 Phalloidin and z stacks were acquired (z step = 1μm). A representative slice from the z stack is shown. Scale bar, 10μm. <b>B.</b> Quantification of number of actin dots formed in Rab21WT and CA, DN mutants when plated on glass or FN coated (100μg/ml) coverslips (n = 150cells). Graph shows mean ± SEM of three independent experiments. **, P<0.01.<b>C.</b>Trophozoites stably expressing HA tagged Rab21CA were harvested and plated on glass and collagen type I (100μg/ml) coated surface and incubated for an hour at 37°C. Cells were fixed and stained for actin using Alexa568 Phalloidin and z stacks were acquired for every z step of 1μm. A representative slice from the z stack is shown. Scale bar, 10μm. <b>D.</b> Quantification of the mean area of actin dots formed in the Rab21CA mutant plated on collagen type I (100μg/ml) coated surface. Mean area was calculated for n = 180 actin dots for collagen type I and n = 145 actin dots for glass surface. Mean diameter was manually measured using the motion tracking software and area calculated, treating the actin dot to be a circular object (</p><p></p><p></p><p><mi>Α</mi><mo>=</mo><mi>π</mi></p><p>r<mn>2</mn></p><p></p><p></p><p></p>) and plotted using GraphPad Prism5. Graph shows mean ± SEM.***, P<0.001. <b>E.</b> Trophozoites transfected with Rab21CA were placed between two layers of either Matrigel at 1mg/ml or collagen type I at 2mg/ml and incubated for 6–8hrs at 37°C. Trophozoites transfected with empty vector were also sandwiched between matrigel as control. Samples were fixed and processed for SEM and imaged using Zeiss Ultra PLUS. Left panel shows lower magnification micrographs (1000X for matrigel, scale bar 10μm and 500X for collagen type I, scale bar 20μm) while the right panel shows higher magnification micrographs (8000X, scale bar 2μm).Arrows indicate surface protrusions produced on Rab21CA cells in a matrigel sandwich compared to the rough surface in a collagen type I sandwich.<p></p

Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in <i>Entamoeba histolytica</i>: Implications in Pathogen Invasion

<div><p>The protozoan parasite <i>Entamoeba histolytica</i> causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with <i>in vitro</i> invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.</p></div

Gal/GalNAc lectin complex inhibits actin dot formation in Rab21CA.

<p>Trophozoites stably expressing Rab21CA were incubated for an hour with <b>A.</b> GalNAc on glass and fixed and stained for actin. <b>B.</b> Quantification of mean area of actin dots in Rab21CA formed on glass surface in presence of 100mM maltose and 100mM GalNAc. Mean diameter was manually measured using the motion tracking software (n = 150 actin dots/condition, total cells counted = 100) and area calculated, assuming the actin dot to be a circular object </p><p></p><p></p><p><mi>Α</mi><mo>=</mo><mi>π</mi></p><p>r<mn>2</mn></p><p></p><p></p><p></p>) and plotted using GraphPad Prism5. Graph shows mean ± SEM.***, P<0.001.<b>C.</b> Rab21 CA transformants were incubated on FN coated surface along with GalNAc for an hour at 35°C. Cells were fixed and stained for actin. <b>D.</b> Quantification of mean area of actin dots in Rab21CA formed on FN coated surface in presence of 100mM maltose and 100mM GalNAc (n = 690 actin dots/ 150 cells for 0mM GalNAc; n = 441 actin dots/ 180 cells for 100mM GalNAc; n = 700actin dots/ 220 cells for 100mM Maltose).Graph shows mean ± SEM.*, P<0.05. <b>E.</b> Similarly, trophozoites stably expressing Rab21CA were incubated with αHgl mouse MAb 3F4 (1:30) plated on glass for an hour at 35°C. Cells were fixed and stained for Hgl (green) and actin (red) and imaged using Zeiss LSM780. <b>F.</b> Quantification of mean area of actin dots in Rab21CA formed on glass surface in presence of αHgl 3F4 (n = 155 actin dots, 91 cells for treated with 3F4; n = 200actin dots, 100cells for untreated).Graph shows mean ± SEM. **, P<0.01. <b>G.</b> Trophozoites stably expressing Rab21CA were incubated with αHgl mouse MAb 3F4 (1:30) plated on fibronectin coated surface for an hour at 35°C. Cells were fixed and stained for Hgl (green) and actin (red) and imaged using Zeiss LSM780. <b>H.</b> Quantification of mean area of actin dots formed on fibronectin coated surface in presence of αHgl 3F4 (n = 590 actin dots, 105 cells for treated with 3F4; n = 450 actin dots, 98 cells for untreated). Graph shows mean ± SEM. *, P<0.05. Scale bars, 10μm.<p></p

Host ECM components induces actin rearrangement in <i>Entamoeba histolytica</i>.

<p><b>A.</b> Control trophozoites were incubated for an hour on Glass and various ECM coated surfaces; Matrigel (100μg/ml); Fibronectin (100μg/ml) and Collagen type I (100μg/ml) at 35°C. Cells were fixed and stained for actin using Alexa568 Phalloidin and imaged using Zeiss LSM 780. <b>B.</b> An orthogonal view of the cells forming actin dots with mean area of 2.5±0.21μm² and a depth of 2–3μm. A representative xy section together with selected xz and yz is shown. For calculation of mean area of actin dots formed in presence of FN, a total of 140 actin dots spread over 70 cells were analyzed. Scale bars, 10μm.</p

Schematic representation of regulation of amoebic actin dots.

<p>The <i>Eh</i>FNR bound to the fibronectin, turns on an intracellular signaling cascade. Rab21 acts downstream of the <i>Eh</i>FNR, possibly by sensing the ligated form of the receptor, a function dependent on its nucleotide bound state. These signaling events finally lead to changes in the actin cytoskeleton of the pathogen. Collagen type I possibly act through the CRD region of the Hgl subunit of the Gal/GalNAc lectin complex and inhibits the amoebic actin dots.</p

Luminescent Copper Nanoclusters as a Specific Cell-Imaging Probe and a Selective Metal Ion Sensor

Copper nanoclusters (CuNCs) exhibit a high tendency to undergo oxidation particularly at the subnanometer size regime. In the light of overcoming this bottleneck, we have been successful in developing tripeptide (glutathione, GSH) templated CuNCs which show high biocompatibility and stability, in spite of being ultrafine in size. These blue-emitting CuNCs possess very promising optical features such as significant quantum yield (QY) and excellent photostability. Our cell-imaging studies reveal that the CuNCs localize primarily in nuclear membranes of the different cancerous (Hela, MDAMB-231, and A549) cells, and the cell viability assay conclusively established their nontoxic nature. Apart from their biological significances, these CuNCs also illustrate their ability to serve as a metal ion sensor, selectively detecting Fe³⁺ ions in solution at the nanomolar concentration regime. This unique luminescent property of the NCs will enable them to serve as label-free and versatile probes having several biological and analytical applications

Insights into the GTP/GDP Cycle of RabX3, a Novel GTPase from <i>Entamoeba histolytica</i> with Tandem G‑Domains

Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from <i>Entamoeba histolytica</i>, <i>Eh</i>RabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that <i>Eh</i>RabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from <i>Eh</i>RabX3 is independent of Mg²⁺, perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that <i>Eh</i>RabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, <i>Eh</i>RabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily