lentivirus encoding egfp pwptorlifeact rfp ires eb3 yfp  (ATCC)

Journal: The Journal of Neuroscience

Article Title: STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones

doi: 10.1523/JNEUROSCI.2496-18.2019

Figure Lengend Snippet: STIM1 regulates microtubule tip dynamics at the periphery of sensory growth cones. A–C, Super-resolution images of growth cones in the absence or presence of the SERCA inhibitor, thapsigargin (200 nm for 10 min) and immunostained for β-III tubulin (cyan), STIM1 (red), or EB3 (green) protein. STIM1 colocalizes with EB3 proteins and microtubules with STIM1/EB3 puncta detected on microtubules in filopodia (A, arrowheads in insets i and ii shown in Ci and Cii, respectively). When growth cones were treated with thapsigargin (B), there was uncoupling of STIM1 and EB3 puncta on microtubules in the peripheral (transition) zone of growth cones (B, inset iii shown in Ciii). D, Colocalization of STIM1/EB3 was greater in the periphery of growth cones in controls (n = 25) compared with growth cones treated with thapsigargin (n = 24, ****p = 8.9E-10, Mann–Whitney U test). Scale bars: A, B, 5 μm; C, 1 μm. E, F, Microtubule tip dynamics were examined in STIM1-CTRL and STIM1-KD growth cones transfected with EB3-YFP. Composite representation of EB3-YFP dash trajectories in (E) STIM1-CTRL and (F) STIM1-KD growth cones, pseudo-colored for dash velocity (μm/s), comprising 5–7 dashes from each of 3 STIM1-CTRL and STIM1-KD growth cones (see also Movies 1, 2; Table 1). Heat map in E represents EB3-dash velocity. G, Total number of filopodia in STIM1-CTRL was greater after EB3-YFP overexpression (n = 12) compared with STIM1-CTRL alone (n = 32, **p = 0.002) or STIM1-KD (n = 30, *p = 0.006) or STIM1-KD EB3-YFP (n = 17, *p = 0.0215). The number of filopodia was not significantly different between STIM1-CTRL growth cones and STIM1-KD (p = 0.621) or STIM1-KD EB3-YFP (p = 0.221), and there was no difference between STIM1-KD and STIM1-KD EB3-YFP (p = 0.435, one-way ANOVA, Tukey's multiple comparison test). H, The number of filopodia with EB3 dashes was reduced in STIM1-KD (n = 17) compared with STIM1-CTRL growth cones (n = 12, **p = 0.0036, Student's t test). I, STIM1-CTRL and (J) STIM1-KD growth cones immunolabeled with drebrin (green) and STIM1 (red). Arrowheads indicate significant drebrin expression at lamellipodial border. Scale bar, 5 μm. K, Drebrin immunoreactivity was increased across whole growth cones after STIM1-KD (n = 25) compared with STIM1-CTRL (n = 21, *p = 0.016, Mann–Whitney U test). L, Drebrin expression in filopodia was inversely correlated to STIM1 expression. The number of filopodia immunoreactive for drebrin was increased after STIM1-KD (n = 25) compared with STIM1-CTRL (n = 21, *p = 0.0162, Student's t test).

Article Snippet: EB3-YFP was transfected using nucleofection (Lonza Walkersville) or with magnetic particles following the manufacturer's instructions (NeuroMag; catalog #NM50200, Oz Biosciences) or subcloned into BamHI and NheI restriction sites of a second-generation lentiviral construct containing the neuron-specific hSyn promoter.

Techniques: MANN-WHITNEY, Transfection, Over Expression, Immunolabeling, Expressing

Journal: The Journal of Neuroscience

Article Title: STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones

doi: 10.1523/JNEUROSCI.2496-18.2019

Figure Lengend Snippet: STIM1 knockdown disrupts EB3 comet-like dash dynamics a

Article Snippet: EB3-YFP was transfected using nucleofection (Lonza Walkersville) or with magnetic particles following the manufacturer's instructions (NeuroMag; catalog #NM50200, Oz Biosciences) or subcloned into BamHI and NheI restriction sites of a second-generation lentiviral construct containing the neuron-specific hSyn promoter.

Techniques:

Journal: The Journal of Neuroscience

Article Title: STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones

doi: 10.1523/JNEUROSCI.2496-18.2019

Figure Lengend Snippet: STIM1 is required for microtubule-ER remodeling and calcium signals in filopodia. A–F, STIM1-CTRL and STIM1-KD neurons were cotransfected with BiP-mCherry-KDEL (ER membrane marker) and EB3-YFP. Images of (A) STIM1-CTRL and (B) STIM1-KD growth cones expressing KDEL (red) and EB3 (green). Scale bar, 5 μm. Ci, Di, KDEL and EB3 localization in single filopodia (outlined). Cii, Dii, Representative kymographs of KDEL (red) and EB3 (green) protrusion into STIM1-CTRL (C) and STIM1-KD (D) filopodia, displayed over 20 and 40 s, respectively. Scale bars, 500 nm. E, The percentage of filopodia with a KDEL signal in STIM1-CTRL filopodia (n = 7) was not significantly different from STIM1-KD filopodia (n = 12) (p = 0.0224, Kruskal–Wallis test). F, The distance EB3 dashes with KDEL signals protruded into filopodia was significantly reduced in STIM1-KD compared with STIM1-CTRL filopodia (***p = 0.003, Kruskal–Wallis test, see Movies 3, 4, 5, 6). G, H, STIM1-CTRL (n = 12) and STIM1-KD (n = 16) sensory neurons were cotransfected with ER-GCaMP6–150 (Ca2+ER reporter) and EB3-tdTomato. Gi, Hi, ER-GCaMP6 (green) and EB3 (red) in filopodia (outlined). Gii, Hii, Representative kymographs of ER-GCaMP6 (green) and EB3 (red) protrusion into STIM1-CTRL (Gii) and STIM1-KD (Hii) filopodia, displayed over 20 and 40 s, respectively. Scale bars, 500 nm. I–M, STIM1-CTRL and STIM1-KD sensory neurons were cotransfected with BiP-mCherry-KDEL and ER-GCaMP6. Images of (I) STIM1-CTRL and (J) STIM1-KD growth cones expressing KDEL (red) and ER-GCaMP6 (green). Scale bar, 5 μm. Ki, Li, ER-GCaMP6 (green) and KDEL (red) in filopodia (outlined). Kii, Lii, Representative kymographs of ER-GCaMP6 (green) and KDEL (red) as ER membrane extends into STIM1-CTRL(Kii) and STIM1-KD (Lii) filopodia, displayed over 20 and 40 s, respectively. Scale bar, 500 nm. M, The filopodial ER-GCaMP6 signal, ΔF/F normalized to KDEL signal, and averaged over 20 s was significantly reduced in STIM1-KD (n = 15) compared with STIM1-CTRL filopodia (n = 16, ****p = 0.00003, Student's t test, see Movies 7, 8).

Article Snippet: EB3-YFP was transfected using nucleofection (Lonza Walkersville) or with magnetic particles following the manufacturer's instructions (NeuroMag; catalog #NM50200, Oz Biosciences) or subcloned into BamHI and NheI restriction sites of a second-generation lentiviral construct containing the neuron-specific hSyn promoter.

Techniques: Marker, Expressing

Journal: The Journal of Neuroscience

Article Title: STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones

doi: 10.1523/JNEUROSCI.2496-18.2019

Figure Lengend Snippet: STIM1 regulates EB3-YFP recruitment to the motile side of turning growth cones. A–D, Maximum intensity projections of EB3 dash trajectories in representative STIM1-CTRL and STIM1-KD growth cones turning to (A,B) BDNF and (C,D) sema-3a (gradient direction demarcated by arrow in top-left corner). EB3-labeled tracks quantified in time over 12 min (color-coded for average velocity per dash, μm/s). E–H, Polar plots depicting EB3 dash displacement and trajectories in STIM1-CTRL (n = 3; E,G) and STIM1-KD (n = 6; F,H) growth cones turning in response to a gradient of (E,F) BDNF or (G,H) sema-3a. Positive angles represent attraction and negative angles represent repulsion with respect to the initial trajectory of the growth cone. I, Near/far ratio of all EB3-YFP dashes in STIM1-CTRL (n = 6) and STIM1-KD (n = 5) growth cones exposed to BDNF or sema-3a gradients (n = 5 STIM1-CTRL and n = 6 STIM1-KD). The final EB3 positions switched from near to far in growth cones turning in response to BDNF after STIM1 knockdown (**p = 0.0033). The final EB3 positions switched from far to a random distribution in growth cones turning in response to sema-3a after STIM1 knockdown (***p = 0.0002, one-way ANOVA, Tukey's multiple comparison test). Inset, Schematic illustrating definition of near (N) and far (F) side of the growth cone with respect to the source of guidance cue (see Movies 9, 10, 11, 12). J, Average turning angles of STIM1-CTRL and STIM1-KD growth cones exposed to BDNF or sema-3a gradients for 12 min (datasets include all growth cones depicted in A–H). Turning angles switched from attraction to repulsion in growth cones turning to BDNF after STIM1 knockdown (**p = 0.006), and from repulsion to random growth in response to sema-3a (**p = 0.008, one-way ANOVA, Tukey's multiple comparison test).

Article Snippet: EB3-YFP was transfected using nucleofection (Lonza Walkersville) or with magnetic particles following the manufacturer's instructions (NeuroMag; catalog #NM50200, Oz Biosciences) or subcloned into BamHI and NheI restriction sites of a second-generation lentiviral construct containing the neuron-specific hSyn promoter.

Techniques: Labeling

Journal: Developmental cell

Article Title: The HCMV Assembly Compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread

doi: 10.1016/j.devcel.2018.03.010

Figure Lengend Snippet: (A) NHDFs were treated with siRNA 30h prior to infection (+) or with a second siRNA treatment at 3d.p.i. (++). Cultures were infected at MOI 3 for 5d. Lysates were analyzed by WB for the indicated proteins. Note, IE1/2 are expressed because early infection is not affected using this siRNA treatment strategy. (B–D) NHDFs were treated with siRNAs as in A. and infected with TB40/E-eGFP at MOI 0.001 for 14d. (B) Cells were lysed and analyzed by WB for the indicated proteins. Note, IE1/2 are reduced because of reduced HCMV spread in EB1 or EB3 depleted cultures. (C) Phase and fluorescent images of plaques for TB40/E or AD169. (D) Plaque sizes in C for TB40/E were measured. n = 3 (>22 plaques); bars = s.e.m. ***p=0.0005, unpaired two-tailed t-test. (E) NHDFs were infected with TB40/E at MOI 3. Cell supernatants and cell-associated virus were harvested at 7d.p.i. and titrated on NHDFs. n = 3; bars = s.e.m; *p<0.05, **p<0.01, unpaired two-tailed t-test. (F) Still images (Movie S5A) of NHDFs expressing eGFP-CLIP170, treated with siRNAs as in A and infected with TB40/E-UL32-mCherry at MOI 1 for 5d. Red boxes highlight elongated eGFPCLIP170 tracks in EB1-depleted cells. (G) Line-scan analysis of eGFP-CLIP170 intensity distribution from the microtubule plus-end in F. n = 10 (>150 MT linescans); bars = s.e.m.

Article Snippet: Preparation of (His6)-YFP-EB3, (His6)-CFP-EB3, (His6)-EB3 and EB3-C terminus (200–281aa) was described previously ( Geyer et al., 2015 ; Komarova et al., 2012 ). (His6)-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) (Stratagene).

Techniques: Infection, Two Tailed Test, Expressing

Journal: Developmental cell

Article Title: The HCMV Assembly Compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread

doi: 10.1016/j.devcel.2018.03.010

Figure Lengend Snippet: (A–D) NHDFs were treated with siRNAs and infected with TB40/E at MOI 1 for 5d. (A) Fixed samples were stained for acetylated tubulin and TGN46. Nuclei were stained with Hoechst. (B) Percentage of cells containing low (as in EB3 siRNA panels in A.), medium (as in Ctrl siRNA panels) or high (as in EB1 siRNA panels) levels of acetylated MTs. n = as indicated. (C) Fixed samples were stained for EB1, EB3 and TGN46. Enlarged insets show EB1 and EB3 comets. (D) Line-scan analysis of EB1 or EB3 comet intensity and distribution in samples in C. Note, loss of one EB increases MT tip binding by the other. Top: Distributions of EB1 (red) or EB3 (green) in control siRNA (solid) or EB1 siRNA (dashed) samples. Bottom: Distributions of EB1 (red) or EB3 (green) in control siRNA (solid) or EB3 siRNA (dashed) samples. n = 5 (>125 MT linescans); bars = s.e.m. (E–F) NHDFs were treated with siRNAs and infected with TB40/E-UL99-eGFP at MOI 0.5. (E) Effects on nuclear rotation: Cells were imaged at 2 frames per hour between 3–5d.p.i. Time lapse images (Movie S5B) were used to measure nuclear rotation. Bottom: Examples of rotations including changes in direction (red and orange). (F) Effects on AC structure: Still images (Movie S6) from faster frame rate analysis of UL99-eGFP showing different AC architectures in control versus EB1 or EB3-depleted cells. Insets use non-linear scaling to show details within the bright AC region, and highlight cytoplasmic virions and MVBs.

Article Snippet: Preparation of (His6)-YFP-EB3, (His6)-CFP-EB3, (His6)-EB3 and EB3-C terminus (200–281aa) was described previously ( Geyer et al., 2015 ; Komarova et al., 2012 ). (His6)-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) (Stratagene).

Techniques: Infection, Staining, Binding Assay

Journal: Developmental cell

Article Title: The HCMV Assembly Compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread

doi: 10.1016/j.devcel.2018.03.010

Figure Lengend Snippet: (A–B) NHDFs treated with DMSO or 25µM HEBTRON or MutN-MutC were infected at MOI 1 for 5d. (A) Fixed cells were stained for TGN46 and CDK5RAP2. Example of measurements of CDK5RAP2-positive area (based on fluorescence above intensity threshold). (B) Area measurements of CDK5RAP2 at the AC above threshold intensity. n = 3 (>90 cells), bars = s.e.m; **p<0.01, unpaired two-tailed t-test. (C) HEBTRON does not affect CDK5RAP2 or EB3 abundance. NHDFs treated with DMSO or 25µM HEBTRON, MutN or MutN-MutC were infected with AD169 at MOI 3 for 5d. Lysates were analyzed by WB. (D–E) NHDFs treated with DMSO or 25µM HEBTRON were infected with TB40/E-UL99-eGFP at MOI 0.5. (D) Time lapse imaging was performed 3–5d.p.i (Movie S7A) and nuclear displacement was measured. Representative traces are shown. (E) Time lapse imaging of the AC at 5d.p.i. (Movie S7B). Still images show AC structure in DMSO or HEBTRON-treated cells. Insets and non-linear scaling show details within the bright AC. Arrows indicate virus particles and MVBs. (F–G) NHDFs treated with DMSO or 25µM HEBTRON were infected with TB40/E at the indicated MOI. Infectious virus in culture supernatants was titrated. (F) Spreading assay infections at MOI 0.001 for 12d. n = 2; bars = s.e.m. (G) Single cycle infections at MOI 1 for 7d. n = 3; bars = s.e.m. *p<0.05, unpaired two-tailed t-test.

Article Snippet: Preparation of (His6)-YFP-EB3, (His6)-CFP-EB3, (His6)-EB3 and EB3-C terminus (200–281aa) was described previously ( Geyer et al., 2015 ; Komarova et al., 2012 ). (His6)-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) (Stratagene).

Techniques: Infection, Staining, Fluorescence, Two Tailed Test, Imaging

Journal: Developmental cell

Article Title: The HCMV Assembly Compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread

doi: 10.1016/j.devcel.2018.03.010

Figure Lengend Snippet: (A) Growth-arrested NHDFs were mock-infected or infected with TB40/E at MOI 3 for the indicated times. Cell lysates were analyzed by WB. (B) Densitometry analysis of EB1, EB2 and EB3 relative to α-tubulin. n = 3; bars = s.e.m.;, *p<0.05, unpaired two-tailed t-test (C) Growth-arrested NHDFs were mock-infected or infected at MOI 3 for 4d. EB transcript levels were measured using qRT-PCR. EB levels were normalized to uninfected controls (arbitrarily set to 1). n = 3; bars = s.e.m; *p<0.05, ***p<0.001, unpaired two-tailed t-test. (D) NHDFs were treated with control or IE1/2 siRNA prior to infection with AD169 at MOI 3 for 3d. Cell lysates were analyzed by WB for the indicated proteins. (E) NHDFs were transduced with retroviruses encoding GFP or IE proteins. Samples were analyzed by WB. (F) NHDFs were infected with AD169 at MOI 3 in the presence of DMSO or CDK1 inhibitor (JNJ-770662), re-dosing daily, for the indicated times. Samples were analyzed by WB.

Article Snippet: Preparation of (His6)-YFP-EB3, (His6)-CFP-EB3, (His6)-EB3 and EB3-C terminus (200–281aa) was described previously ( Geyer et al., 2015 ; Komarova et al., 2012 ). (His6)-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) (Stratagene).

Techniques: Infection, Two Tailed Test, Quantitative RT-PCR, Transduction

Journal: Developmental cell

Article Title: The HCMV Assembly Compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread

doi: 10.1016/j.devcel.2018.03.010

Figure Lengend Snippet: (A) ITC of HEBTRON binding to purified EB3 C-terminus (200–281aa). KD, binding enthalpy, and stoichiometry were calculated from changes in heat upon binding of HEBTRON to the protein using the "one set of sites" binding model. (B) Thermal unfolding of full-length EB3 alone (blue) or with HEBTRON (red) using NanoDSF. The first derivative of the 350/330 nm (peak) defines transitions between folded (left) to unfolded (right) states. Magnified insets show 0.2°C temperature shift (green lines) for the complex. This suggests HEBTRON stabilizes dimers. (C) FRET signal after mixing full-length EB3-CFP and EB3-YFP without (blue) or with HEBTRON (red). FRET indicates formation of YFP/CFP-EB3 dimers, blocked by HEBRTON at 1:1 molar ratio. (D) NHDFs were treated with 10µM Myr-FITC-conjugated HEBTRON. Time lapse images were taken and overlaid with DIC images at the indicated times. (E) % uptake of Myr-FITC-conjugated HEBTRON in NHDFs over time was determined by the normalized fluorescence intensity of the cell. n = 3; assaying 32 cells. Bars = s.e.m. (F) NHDFs treated with DMSO or 25µM HEBTRON were infected with TB40/E at MOI 1 for 5d. Fixed cells were stained for acetylated tubulin and gB. (G) % DMSO or HEBTRON-treated cells in F containing acetylated MTs. n = as indicated. (H) Sequence of HEBTRON, MutN or MutN-MutC. (I) NHDFs treated with DMSO or 25µM HEBTRON, Mut-N or MutN-MutC were infected with TB40/E-eGFP at MOI 0.001 for 12d. Representative phase and fluorescent images of plaques. (J) Cells were treated and infected with TB40/E or AD169 as in I. Plaque areas are shown. n = 3 (>40 and >50 plaques. respectively); bars = s.e.m.; p*<0.05, **p<0.01, ***p<0.001, unpaired two-tailed t-test.

Article Snippet: Preparation of (His6)-YFP-EB3, (His6)-CFP-EB3, (His6)-EB3 and EB3-C terminus (200–281aa) was described previously ( Geyer et al., 2015 ; Komarova et al., 2012 ). (His6)-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) (Stratagene).

Techniques: Binding Assay, Purification, Nano Differential Scanning Fluorimetry, Fluorescence, Infection, Staining, Sequencing, Two Tailed Test