Tim is a c-Src SH3 domain binding protein and substrate.

<p><b>A</b>, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. <b>B</b>, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.</p

Tim knockdown prevents EB cavitation.

<p>EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 days. Fixed EBs were then stained with DAPI (nuclei; blue) plus Alexa Fluor 488-phalloidin (F-actin; green) and imaged by confocal microscopy using an Olympus Fluoview 1000 confocal microscope. <b>A</b>, Optical sections (5 µm) were taken from the bottom through the middle of the EB where cavitation is the greatest. Images of representative sections are shown of the bottom as well as one-quarter and halfway through the EB. <b>B</b>, Side profiles from merged images reveal cavitation (“c”) in the control but not the Tim knockdown EBs. <b>C</b>, Bargraph shows the percentage of cavitated EBs formed from the parental mES cell line, mES cells transduced with a nontargeted shRNA lentivector (Non-T) as well as the two Tim knockdown ES cell lines.</p

Timeless-knockdown EBs are resistant to apoptosis.

<p><b>A</b>, Control and Tim-knockdown EBs were grown for 6 days from the corresponding ES cell lines and incubated in the presence or absence of 0.5 µM staurosporine for 16 h. Cell lysates were analyzed for Caspase 3/7 activity using the Apo-1 assay. The data are normalized to the activity observed with control EBs in the presence of staurosporine. The experiment was repeated three times, and the results are presented in the bargraph as mean percent of control ± S.E.M. <b>B</b>, Lysates from staurosporine-treated control and Tim-knockdown EBs were analyzed for the presence of the active form of Capsase-3 by immunoblotting with an antibody specific for the cleaved, active form of this protease. Signal intensities were quantified using the LI-COR Odyssey system, and normalized to control values. The experiment was repeated in triplicate, and the bargraph shows the mean values ± S.E.M. EBs derived from both Tim knockdown cell lines showed a significantly reduced apoptotic response to staurosporine treatment in each of these assays (p≤0.02 in each case).</p

Generation of Tim knockdown ES cell lines.

<p>Endogenous Tim expression was suppressed in mES cells by transduction with lentiviral particles carrying shRNA sequences targeting independent regions of the <i>Tim</i> locus (Lenti:87 and Lenti:89). Following puromycin selection, 12 undifferentiated colonies were picked, expanded, and the levels of Tim protein expression determined by quantitative immunoblotting. <b>A</b>, Morphology of representative Tim-knockdown lines isolated from the Lenti:87 and Lenti:89 ES cell populations. Control ES cell colony morphology is also shown for comparison. <b>B</b>, The relative level of full-length Tim in lysates from each of the Tim knockdown lines shown in Part <b>A</b> was determined by quantitative immunoblotting (Tim; arrow). Immunoblots were also probed with an actin antibody as a loading control, and the relative levels of each protein were quantitated using the LI-COR Odyssey system and secondary antibodies conjugated to infrared fluorphores. <b>C</b>, Bargraph showing the Tim:actin protein ratios. Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 were used in subsequent experiments based on unchanged ES cell colony morphology (Part A; red outline) and extent of Timeless knockdown.</p

EBs formed from Tim knockdown cells retain pluripotent cells.

<p>EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. <b>A</b>, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. <b>B</b>, Side profiles from merged images in part <b>A</b>; the location of the cavity in the control EB is indicated with a “c”. <b>C</b>, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. <b>D</b>, Secondary EB assay. Six-day EBs from part <b>A</b> were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).</p

Tim knockdown EBs are smaller and of more uniform size.

<p>EBs were grown from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. EBs were fixed, DAPI-stained and imaged with an Olympus F500 confocal microscope. The EB sizes were then estimated by determining the EB surface area from the images using the <u>ImageJ 1.43U software</u> suite. Scattergrams of the resulting data were analyzed using the Kruskal-Wallis test (nonparametric unpaired analysis of variance by ranks; Prizm Software, GraphPad, Inc.) and a significant difference in the median EB size was observed across both groups (P<0.0001). The median EB size is shown in each group by the red bar.</p

Changes in Tim protein levels and tyrosine phosphorylation during EB formation.

<p><b>A and B</b>, Tim protein levels diminish during EB formation. Lysates were prepared from self-renewing ES cells (ESC) and 3, 6, and 12 day EBs, and immunoblotted for Tim and actin protein levels. Full length Tim as well as two possible cleavage products (CP) are indicated by the arrows. Relative band intensities for full-length Tim and actin were determined using ImageJ from four independent experiments and Tim:actin ratios were calculated. The results were normalized to ratios obtained from control ES cells, and are presented in the bargraph as the mean ± S.E.M. The level of Tim was significantly reduced after 6 and 12 days of EB formation (p≤0.01). <b>C</b>, Tyrosine phosphorylation of endogenous Tim in ES cells and EBs. Lysates were prepared from self-renewing ES cells and 3, 6, and 12 day EBs, and tyrosine-phosphorylated proteins were immunoprecipitated from protein aliquots and analyzed for Tim by immunoblotting (top panel). Actin blots were performed to verify equivalent levels of input protein for the immunoprecipitation (lower panel). This experiments was repeated three times with comparable results; a representative example is shown. <b>D</b>, Inhibition of Tim tyrosine phosphorylation in ES cells by Src-family kinase inhibitors. ES cells were incubated with the Src-family kinase inhibitors PP2 and SKI-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017157#pone.0017157-Meyn1" target="_blank">[12]</a> at 10 µM for 16 h. Tyrosine-phosphorylated proteins were immunoprecipitated and analyzed for the presence of Tim by immunoblotting (top panel). Tim blots (lower panel) verified equivalent levels of Tim in each lysate prior to immunoprecipitation.</p

Knockdown of Tim suppresses spontaneous differentiation of mES cells.

<p><b>A</b>, Representative images of control and Tim knockdown ES cell lines 87-22 and 89-18 after 24 and 48 h in culture. Note that spontaneously differentiating mES cell clusters are readily apparent in control ES cell cultures by 48 h (flat colonies with ragged edges; “<i>d</i>”) but are absent from the Tim knockdown cultures. <b>B</b>, Analysis of self-renewal and differentiation marker expression in Tim knockdown cell lines. Expression levels of the self-renewal markers Oct4, Sox2, Nanog, KLF4, the differentiation marker AFP, as well as Tim were assessed by quantitative immunoblotting (LI-COR Odyssey infrared imaging system) of cell lysates from parental ES cells as well as the Tim knockdown ES cell lines 87-22 and 89-18. Actin immunoblots served as loading control. Immunoblots were performed in triplicate and the level of each protein was normalized to actin and is shown in the bargraph as the mean ± S.E.M. Sox2 expression levels were significantly increased in the Tim knockdown cells relative to parental ES cells (p<0.05), while AFP showed a statistically significant decrease (p<0.05). While small increases in Oct4 expression were also observed in the Tim knockdown cell lines, these changes were not statistically significant.</p

Using total internal reflection fluorescence (TIRF) microscopy to visualize cortical actin and microtubules in the Drosophila syncytial embryo.

The Drosophila syncytial embryo is a powerful developmental model system for studying dynamic coordinated cytoskeletal rearrangements. Confocal microscopy has begun to reveal more about the cytoskeletal changes that occur during embryogenesis. Total internal reflection fluorescence (TIRF) microscopy provides a promising new approach for the visualization of cortical events with heightened axial resolution. We have applied TIRF microscopy to the Drosophila embryo to visualize cortical microtubule and actin dynamics in the syncytial blastoderm. Here, we describe the details of this technique, and report qualitative assessments of cortical microtubules and actin in the Drosophila syncytial embryo. In addition, we identified a peak of cortical microtubules during anaphase of each nuclear cycle in the syncytial blastoderm, and using images generated by TIRF microscopy, we quantitatively analyzed microtubule dynamics during this time.</p

Endocytosis of KCa2.3 is dependent upon dynamin.

<p><b>A.</b> GFP-tagged WT dynamin II and BLAP-tagged KCa2.3 were expressed in HEK293 cells. KCa2.3 was labeled at the plasma membrane with streptavidin-Alexa555 for 10 min at 4°C after which the cells were immediately imaged by live-cell confocal microscopy at 37°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044150#s2" target="_blank">Methods</a>). <b>Left Panels</b> show individual images from two separate cells during the time course of the experiment. <b>Right Panels</b> show cropped images from these two cells (labeled 1 and 2) as well as cropped images from 2 additional cells. Arrows denote co-localization of KCa2.3 and WT dynamin II. <b>B.</b> Co-IP of myc-tagged KCa2.3 with GFP-tagged dynamin II. KCa2.3 was immunoprecipitated using an anti-myc Ab (lanes 1, 2, 4, 5) or an anti-V5 Ab as IgG control (lane 3) and subsequently IB using an anti-GFP Ab for dynamin II. WT and K44A dynamin II were detected by IB in lanes 4 and 5, respectively, confirming an association between KCa2.3 and dynamin (Top Panel). Bottom Panel confirms expression of GFP-tagged dynamin in total lysate by IB (5 µg total protein loaded per lane). Data are representative of 3 experiments.</p