Datasets:

TomTBT

/

pmc_open_access_figure_noncomm

like 0

PMC1351290

15753208

20041470f6

Figure 6. In vitro cytokine production. (A) Lesion-derived L. major amastigotes were added to bone marrow–derived macrophages in the presence of inflammatory LMW-HA. Supernatants were harvested 24 h later and cytokines IL-12 (gray bars) and IL-10 (black bars) were measured by ELISA. (B) The production of cytokines, IFN-γ and IL-4, from T cells was measured by ELISA 3 d after primary stimulation. OVA-specific TCR transgenic T cells were added to either uninfected (white bars) or L. major –infected (black bars) macrophages cultivated with OVA and LMW-HA.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f7

Figure 7. Flow cytometry to detect intracellular IL-10. Human monocytes were infected with a 10:1 ratio of axenic amastigotes of L. chagasi . Before infection, some amastigotes were incubated at 4°C for 15 min in 5% serum from either an uninfected volunteer (naive serum) or from a patient with visceral leishmaniasis (VL serum). Monocytes were incubated in Golgi stop for 2 h, fixed, and permeabilized and stained for intracellular IL-10 expression. Cells were gated on CD14 expression.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f8

Figure 8. DTH and antibody responses in patients with visceral leishmaniasis (VL). Delayed-type hypersensitivity (DTH) reactions were measured in 317 bone marrow aspirate-confirmed VL patients. The mean induration size of the DTH response (in mm diameter) was measured at 48 h. (inset table) The same VL patients were analyzed for antibody and DTH responses. Antibody levels were judged to be positive if they were >3 SD above a mean control titer. DTH responses >5 mm in diameter were judged to be positive. This figure designates a significant negative association between DTH and antibody responses. Pearson χ 2 , df = 1; P ≤ 0.0001.

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351290

15753208

20041470f9

Figure 9. DTH and Leishmania-specific antibody responses in VL patients after treatment. The mean diameter induration score of the DTH response, measured in mm, is shown by the black bars (left), and the mean antibody titers, expressed as OD reading, are shown by the white bars (right) as a function of time after treatment (abscissa).

CC BY-NC-SA

no

2022-01-13 09:00:57

J Exp Med. 2005 Mar 7; 201(5):747-754

PMC1351313

16009725

200504167f1

Figure 1. Regulation of CAPRI by agonist-evoked Ca 2 + signals. (A) Confocal images of HeLa cells expressing GFP-CAPRI (left) or GFP-RASAL (right) before (T = 0) or after 100 μM histamine stimulation. T = time (s). ROI = region of interest used to calculate the Relative Translocation parameter (see Materials and methods). (B) Translocation of GFP-CAPRI after 100 μM (bold trace; average n = 7 cells, n = 6 experiments) or 10 μM histamine (light trace; n = 5 cells, n = 2 experiments). (C) Translocation of GFP-RASAL (green trace; n = 3 cells) and GFP-PKCγ (red trace; average n = 12 cells, n = 2 experiments) after 100 μM histamine.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Jul 18; 170(2):183-190

PMC1351313

16009725

200504167f2

Figure 2. CAPRI is refractory to Ca 2 + oscillations. (A) Ca 2+ mobilization induced by application of 50 μM ATP in CHO cell lines. Black = parental CHO T cell response ( n = 35 cells); blue = CHO-CAP6 clone stably expressing CAPRI ( n = 57 cells); red = CHO-MUT13 clone stably expressing CAPRI (R473S) ( n = 23 cells). (B) Representative trace of the change in Fura-2 emission (black trace) and GFP-CAPRI translocation (blue trace) from a single cell displaying sinusoidal Ca 2+ oscillations after 50 μM histamine. (C) Representative trace of the change in Fura-2 emission (black trace) and GFP-CAPRI translocation (blue trace) from a single cell displaying baseline Ca 2+ spikes after 5 μM histamine. (D) Ca 2+ mobilization in HeLa cells (black trace) after 50 μM histamine with sequential RFP-CAPRI translocation (blue trace, average n = 5 experiments) (E) Reversibility of GFP-CAPRI translocation by histamine. Change in Fura-2 emission indicated in black, relative CAPRI translocation in blue. Cells stimulated with 50 μM histamine for 60 s, followed by wash-out of agonist, followed by restimulation for 60 s as shown (average n = 3 cells). (F) Translocation of CAPRI-YFP and CFP-RASAL induced by 50 μM ATP in HeLa cells.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Jul 18; 170(2):183-190

PMC1351313

16009725

200504167f3

Figure 3. Role of the tandem C2 domain and PH domain of CAPRI. (A) Top: 5 μM ionomycin is sufficient to drive GFP-C2AB CAPRI to the HeLa inner nuclear membrane (+ ion = 20 s after stimulation). Translocation is Ca 2+ -dependent (+ EGTA = 40 s after 5 mM EGTA-containing media). Bottom: GFP-C2AB plasma membrane translocation induced by histamine. (B) GFP-C2AB translocation in HeLa cells stimulated with 100 μM histamine. Bold trace is GFP-C2AB (average n = 7 cell, n = 3 experiments) compared with average GFP-CAPRI translocation (dotted trace) under similar conditions (see Fig. 1 B). (C) Representative response of GFP-CAPRI/RASAL chimera to 10 μM histamine. (D) Response of GFP-RASAL/CAPRI chimera to 100 μM histamine (average n = 6 cells, n = 2 experiments). (E) Response of GFP-CAPRI (W664A) to 100 μM histamine (average n = 12 cells, n = 3 experiments).

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Jul 18; 170(2):183-190

PMC1351313

16009725

200504167f4

Figure 4. TIRFM. (A) GFP-CAPRI translocation induced by 100 μM histamine monitored by TIRFM (representative experiment for n = 4 cells). (B) GFP-RASAL translocation induced by 100 μM histamine monitored by TIRFM (representative experiment for n = 3 cells). (C) Representative experiment of GFP-CAPRI translocation in HeLa cells stimulated with 1 μM histamine ( n = 3 cells). Arrows indicate mini-oscillations in one trace; similar oscillations are also detectable in other cells as shown. (D) Representative trace of sequential Fura-2 (black trace; single wavelength 380-nm excitation) and TIRF imaging of GFP-CAPRI (blue trace). Mini-oscillations correlate with cytosolic Ca 2+ spikes.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Jul 18; 170(2):183-190

PMC1351313

16009725

200504167f5

Figure 5. Ca 2 + -triggered Ras deactivation by CAPRI in live cells. (A) GFP-CAPRI translocation correlates with HcRed-RBD dissociation. HeLa cells cotransfected with GFP-CAPRI, HcRed-RBD, and H-Ras. Time after 50 μM histamine stimulation of nonstarved cells is indicated. Nuclear HcRed fluorescence is masked. To enhance red signal, curves were adjusted simultaneously across the images so that no single image was enhanced over another. (B) ATP-induced deactivation of H-Ras in COS-7 cells cotransfected with CAPRI, GFP-RBD, and H-Ras. Time after 50 μM ATP stimulation of nonstarved cells indicated. Arrows highlight GFP-RBD ruffles. G = Golgi apparatus; inset is Golgi at lower image intensity. The experiment is best viewed in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200504167/DC1 ). (C) Relative dissociation of GFP-RBD by ATP-induced activation of CAPRI in COS-7 cells (black trace). Nonstarved cells were cotransfected with CAPRI, H-Ras, and GFP-RBD, and were stimulated with 50 μM ATP ( n = 9 cells, n = 6 experiments ± SD). Gray trace indicates GFP-RBD dissociation in the absence of CAPRI transfection ( n = 4 cells, n = 2 experiments). (D) Relative dissociation of GFP-RBD by histamine-induced activation of CAPRI in HeLa cells. Nonstarved HeLa cells were cotransfected with CAPRI, H-Ras, and GFP-RBD, and were stimulated by 100 μM histamine ( n = 4 cells, n = 2 experiments ± SD). (E) Comparison of the GFP-CAPRI translocation induced by 50 μM ATP (average n = 8 cells, n = 3 experiments) to dissociation of GFP-RBD from the membrane in C. (F) Comparison of the GFP-CAPRI translocation induced by 100 μM histamine in HeLa cells ( Fig. 1 B) to dissociation of GFP-RBD from the membrane in D.

CC BY-NC-SA

no

2022-01-13 07:26:07

J Cell Biol. 2005 Jul 18; 170(2):183-190

PMC1361263

15096539

20031560f1

Figure 1. DBY elicits a sustained T cell response after allogeneic HSCT. (A) IFN-γ secretion ELISPOT assay was performed using unstimulated PBMCs collected 15 mo after HSCT and 93 overlapping DBY peptides distributed into 12 pools. Results are presented as number of spot-forming cells/10 6 PBMCs. (B) The eight peptides included in pool-1 were tested individually using the same cells and according to the same procedure. (C) Patient PBMCs collected at various times before and after HSCT were tested by ELISPOT assay for reactivity to the DBY antigenic peptide. Time 0 corresponds to a sample collected before HSCT.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f2

Figure 2. The CD4 + T cell response to DBY antigen is restricted by HLA-DRB1*1501 molecules. (A) CD4 + T cell clone, 2F9, was stimulated with autologous EBV-immortalized B cells pulsed with DBY peptide (DBY 30–48 ) or another irrelevant DBY peptide. Anti–HLA class II mAb (9.49) and anti–HLA-DR mAb (G46.6) blocked antigen recognition, whereas anti–HLA class I (W6/32) or anti–HLA-DQ (SPVL.3) mAb did not. Results represent quantitative measurement of IFN-γ secretion in the coculture supernatant obtained by ELISA. (B) Clone 2F9 was stimulated with different DBY 30–48 –pulsed EBV B cells (LCL) sharing the expression of either HLA-DRB1*0301 or HLA-DB1*1501 with the patient cells. After overnight incubation, IFN-γ secretion was measured in the coculture supernatant.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f3

Figure 3. DBX and DBY epitopes are both recognized by patient T cells. (A) 2F9 T cells were stimulated with autologous EBV B cells pulsed with different concentrations of DBX and DBY peptides. IFN-γ secretion was measured in the coculture supernatant after overnight incubation. The optimal 19-mer DBX and DBY peptides are shown in • and ○, respectively. Shorter, 14-mer suboptimal epitopes are also shown (▪ and □). Disparate residues between DBX and DBY versions of the peptides are represented with bold underlined characters. Peptide concentrations are expressed in microgram/milliliter. (B) IFN-γ secretion ELISPOT assays were performed using unstimulated PBMCs collected 8 and 21 mo after HSCT and both DBX 30–48 and DBY 30–48 peptides. (C) Clone 2F9 cytolytic activity was tested against EBV B cells pulsed with either DBX 30–48 (▪), DBY 30–48 (•), or an irrelevant peptide, DBY 536–552 (▴).

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f4

Figure 4. T cell clone 2F9 recognizes DBX and DBY antigens endogenously processed and presented by mature DCs. (A) Female and male HLA-DRB1*1501 DCs (10 4 /well) were incubated for 24 h with medium alone or poly-(I)-poly-(C) to induce maturation. T cell clone 2F9 (10 4 /well) was then added to the wells in the presence of IL-2. After 18 h of incubation, secretion of IFN-γ was determined in cocultures using ELISA. (B) Clone 2F9 cytolytic activity toward DBY 30–48 –pulsed 51 Cr-labeled EBV B cells (LCL) at an E/T ratio of 1:1 was inhibited by the addition of mature unlabeled HLA-DRB1*1501 female or male DCs. Three ratios of unlabeled/labeled targets were tested in this experiment.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f5

Figure 5. DBY antigen elicits an antibody response after HSCT. IgG antibodies to recombinant DBY protein were detected by ELISA in patient serum samples collected at various times after allogeneic HSCT. Results are presented as serum titrations. Nonspecific reactivity to a control recombinant protein (HIV p24) was subtracted from each value.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f6

Figure 6. The antibody response after HSCT is specific for DBY antigen. DBX and DBY recombinant proteins were separated by SDS-PAGE electrophoresis, transferred on nitrocellulose membrane, and immunoblotted with either an anti-V5 mAb (left) or patient serum collected 21 mo after HSCT (right). Degradation products generating distinct patterns for DBX and DBY are noticeable in the figure.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f7

Figure 7. The antibody response to DBY continues to evolve 21 mo after HSCT. (A) Patient serum collected 16 mo after transplant was used to map the DBY-specific antibody response. The serum was diluted at 1:10 and tested by ELISA for reactivity with each of the 93 overlapping DBY peptides. (B) The same experiment was repeated using identical procedures with serum collected 21 mo after HSCT.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361263

15096539

20031560f8

Figure 8. Antibody responses after HSCT are specific for DBY antigen. Patient serum collected 21 mo after HSCT was tested in ELISA for reactivity with two DBY B cell epitopes, the corresponding DBX peptides, and both DBY and DBX T cell epitopes. Peptide sequences are shown with the X-Y–disparate residues noted with bold underlined characters.

CC BY-NC-SA

no

2022-01-13 08:52:51

J Exp Med. 2004 Apr 19; 199(8):1133-1142

PMC1361683

15337779

200406148f1

Figure 1. Diagram illustrating the central pair images expected from longitudinal thin sections through flagella. Thin sections of CP that are parallel to the section plane reveal both CP microtubules. If the CP is perpendicular, the section reveals a single CP microtubule. If the CP is twisted, parallel and perpendicular views alternate and one CP microtubule tracks from one edge to the other edge of the parallel views.

CC BY-NC-SA

no

2022-01-13 07:23:09

J Cell Biol. 2004 Aug 30; 166(5):709-715

PMC1361683

15337779

200406148f2

Figure 2. Central pair orientation in quiescent wild-type flagella. (A) A section perpendicular to the glass surface (dashed line) shows one adherent cell and basal regions of both flagella as they exit the cell wall. (B) Low magnification view parallel to the glass surface shows many pairs of flagella. Arrow in C indicates location of a CP twist, whereas no twists are visible in either flagellum in D. Images in C and D have been distorted by a linear scale transformation in one dimension to emphasize CP orientations; insets in each figure show undistorted images. Bars: (A and insets C and D) 1 μm; (B) 5 μm.

CC BY-NC-SA

no

2022-01-13 07:23:09

J Cell Biol. 2004 Aug 30; 166(5):709-715

PMC1361683

15337779

200406148f3

Figure 3. Central pair orientation in quiescent spoke-defective flagella. Each pair of images includes one undistorted image (A–C) and one image distorted to emphasize CP orientation (A'–C'). (A and A') A pf17 flagellum in which the CP has two twists (arrows). (B and B') A pf1sup-pf1 flagellum with one twist (arrow). (C and C') A curved pf1sup-pf1 flagellum with one twist (arrow) between bends of opposite curvature. CP microtubules are parallel to the substrate in curved regions. Bars, 1 μm.

CC BY-NC-SA

no

2022-01-13 07:23:09

J Cell Biol. 2004 Aug 30; 166(5):709-715

PMC1361683

15337779

200406148f4

Figure 4. Central pair orientation in beating spoke-defective flagella. (A and C) CP orientation (emphasized by parallel white lines in the enlargements) is parallel to the bend plane in principal bends. (B) The CP twists (arrow) in a straight region between principal and reverse bends (image distorted by a linear transformation; inset shows an undistorted image). The 32-nm repeat projection (small arrows) diagnostic for C1 appears along the outer edge of both principal bends (C) and reverse bends (D). Insets show the location of enlarged regions. Bars: (insets) 1 μm; (enlargements) 100 nm.

CC BY-NC-SA

no

2022-01-13 07:23:09

J Cell Biol. 2004 Aug 30; 166(5):709-715

PMC1361683

15337779

200406148f5

Figure 5. Helical shape of extruded CP complexes. (A) CP extruded from wild-type flagella and visualized by dark field microscopy are helical. Two images in the top row show one CP at different focal planes. Black lines indicate the borders of individual images assembled for this panel. (B–D) Helical CP flatten onto specimen grids when negatively stained. Changes in curvature (boxed region in B, enlarged in C) are accompanied by twists (C, arrow). The two CP microtubules are side by side in curved regions (C and D) with C1 (marked by 32-nm repeat projections, small arrows in D) along the outer edge of each curve. A dashed line in C follows the midline of the C1 microtubule as it passes through a twist. Bars: (A) 5 μm; (B) 1 μm; (C and D) 100 nm.

CC BY-NC-SA

no

2022-01-13 07:23:09

J Cell Biol. 2004 Aug 30; 166(5):709-715

PMC1364507

15967825

20042280f1

Figure 1. α -GalCer activates peripheral NK cells. C57BL/6 WT mice were treated on days 0 and 4 i.p. with 1 μg α-GalCer (200 μl). Mice were harvested 1, 2, 3, 7, and 10 d after the second α-GalCer injection. Spleen cells were examined for NK cell activation as defined by surface CD69 expression. Naive WT control mice are indicated by the gray line and treated mice are indicated by the solid lines. Results are representative of two different experiments.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f2

Figure 2. Prolonged and elevated NK cell pfp-mediated cytotoxicity after α -GalCer/IL-21 combination. C57BL/6 WT or gene-targeted mice were treated on days −4 and 0 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle, and on day 3 i.v. with 20 μg of pORF or pIL-21 DNA plasmid. Mice were harvested at various time points, and spleen cells were examined in a 4-h 51 Cr release assay at several effector/target (E:T) ratios using Yac-1 target cells. (A) Cytotoxicity of WT spleen cells from treated mice at 25:1 on various days of harvest and (B) cytotoxicity of WT spleen cells on day 6 at various E:T ratios. Results were expressed as the mean percentage lysis ± SEM of triplicate samples. N.T., not tested.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f3

Figure 3. The role of pfp and IFN- γ in prolonged NK cell cytotoxicity by α -GalCer/pIL-21. C57BL/6 WT or gene-targeted mice were treated on days −4 and day 0 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle, and on day 3 i.v. with 20 μg of pORF or pIL-21 DNA plasmid. Mice were harvested at various time points, and spleen cells examined in a 4-h 51 Cr release assay at several effector/target (E:T) ratios using Yac-1 target cells. (A) Cytotoxicity at 25:1 on days 3, 6, and 10 for spleen cells from WT versus gene-targeted mice. Results were expressed as the mean percentage lysis + SEM of triplicate samples. N.T., not tested. (B) Western analysis of spleen cells harvested from vehicle/pORF-, vehicle/pIL-21–, α-GalCer/pORF–, and α-GalCer/pIL-21–treated WT mice 3 d after the pORF/pIL-21 injection (day 6 above). Immunoblots were probed with mAb to pfp (67 kD) or α-tubulin (50 kD) and visualized by enhanced chemiluminescence. (C) α-GalCer–treated mice were injected with control pORF or pIL-21 vector on day 3 after the second α-GalCer injection. Mice were harvested 3 d after the pORF vector injection, and spleen cells were examined for NK cell surface phenotype and intracellular IFN-γ levels. As internal control, naive B6 mice were injected with poly I:C (200 μg), and spleen cells were harvested 3 h later and subjected to intracellular IFN-γ staining. Results are representative of two different experiments.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f4

Figure 4. α -GalCer/IL-21 combination suppresses tumor metastases and prolongs survival. Groups of five C57BL/6 WT mice were injected i.v. with 5 × 10 5 B16F10 tumor cells. Mice were treated: (A and C) on days 0 (the day of tumor inoculation) and 4 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on day 7 i.v. with 20 μg of pORF or pIL-21 DNA plasmid; (B and D) on days 4 and 8 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on day 11 i.v. with 20 μg of pORF or pIL-21 DNA plasmid; and (E) on days 0 and 4 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on days 4, 7, or 10 i.v. with 20 μg of pIL-21 DNA or pORF (day 7) plasmid as indicated. In some experiments (A, B, and E), 14 d after tumor inoculation the lungs of the mice were harvested, and tumor colonies were counted and recorded as the mean number of colonies ± SEM. Asterisks indicate the groups in which combined α-GalCer/pIL-21 treatment significantly reduced the number of lung metastases compared with all other groups (Kruskal-Wallis: *P < 0.05). In other experiments (C and D), the survival of the mice was monitored and recorded as the mean survival time (days) ± SEM. Asterisks indicate the groups in which combined α-GalCer/pIL-21 treatment significantly increased survival compared with all other groups (Kruskal-Wallis: *P < 0.05). Results are representative of three experiments.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f5

Figure 5. α -GalCer/IL-21 combination suppresses tumor metastases by pfp and IFN- γ -dependent mechanisms. Groups of (A) five C57BL/6 WT and C57BL/6 gene-targeted mice were injected i.v. with 5 × 10 5 B16F10 tumor cells and (B) five BALB/c WT and BALB/c gene-targeted mice were injected with 2.5 × 10 5 Renca tumor cells. Some groups of mice were depleted of NK cells by treatment with rabbit anti-asGM1 antibody on days −1, 0 (the day of tumor inoculation), and 7. Mice were treated: (A) on days 4 and 8 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on day 11 i.v. with 20 μg of pORF or pIL21 DNA plasmid; and (B) on days 0 (the day of tumor inoculation) and 4 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on day 7 i.v. with 20 μg of pORF or pIL21 DNA plasmid. In both experiments (A and B), 14 d after tumor inoculation the lungs of these mice were harvested, and tumor colonies were counted and recorded as the mean number of colonies ± SEM. Asterisks indicate the groups in which combined α-GalCer/pIL-21 treatment did not reduce lung metastases significantly compared with WT mice (Kruskal-Wallis: *P < 0.05).

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f6

Figure 6. α -GalCer/IL-21 combination suppresses spontaneous tumor metastases. Groups of five BALB/c WT mice were injected into the mammary gland with 2.5 × 10 4 4T1 tumor cells. On day 8 primary tumors were resected surgically. Mice were treated on days 10 and 14 i.p. with 1 μg α-GalCer (200 μl) or 200 μl of vehicle and on day 17 i.v. with 20 μg of pORF or pIL21 DNA plasmid. Some groups of mice were killed at day 25 and (A) liver and (B) lung metastases were quantitated as described in Materials and methods. Tumor metastases were counted and recorded as the mean number of metastases ± SEM. Asterisks indicate the groups in which combined α-GalCer/pIL-21 treatment reduced metastases significantly compared with WT mice (Kruskal-Wallis: *P < 0.05). (C) Other identical treatment groups were monitored for survival, and data were recorded as mean survival time (days) ± SEM. Asterisks indicate the groups in which combined α-GalCer/pIL-21 treatment significantly increased survival compared with all other groups (Kruskal-Wallis: *P < 0.05). Results are representative of two experiments.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f7

Figure 7. α -GalCer/DC/IL-21 combination mediates extremely potent suppression of tumor metastases. Groups of five C57BL/6 WT mice were injected i.v. with 5 × 10 5 B16F10 tumor cells. Mice were treated: (A and C) on days 0 (the day of tumor inoculation) and 4 i.v. with 5 × 10 5 α-GalCer–pulsed DCs (+) or vehicle-pulsed DCs (–) and on day 7 i.v. with 20 μg of pORF or pIL21 DNA plasmid; and (B and D) on days 4 and 8 i.v. with 5 × 10 5 α-GalCer–pulsed DCs (+) or vehicle-pulsed DCs (−) and on day 11 i.v. with 20 μg of pORF or pIL21 DNA plasmid; and (E) on days 4 and 8 i.v. with 5 × 10 5 α-GalCer–pulsed DCs (+) or vehicle-pulsed DCs (−) and on days 11, 12, and 13 with 50 μg of recombinant mouse IL21 or PBS. In some experiments (A, B, and E), 14 d after tumor inoculation the lungs of these mice were harvested, and tumor colonies were counted and recorded as the mean number of colonies ± SEM. Asterisks indicate the groups in which combined α-GalCer/DC/IL-21 treatment significantly reduced the number of lung metastases compared with all other groups (Kruskal-Wallis: *P < 0.05). In other experiments (C and D) the survival of these mice was monitored and recorded as the mean survival time (d) ± SEM. Asterisks indicate the groups in which combined α-GalCer/DC/pIL-21 treatment significantly increased survival compared with all other groups (Kruskal-Wallis: *P < 0.05). Results are representative of two experiments.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f8

Figure 8. α -GalCer/DC/IL-21 combination suppresses MCA initiation of sarcoma. Groups of 10 C57BL/6 WT mice were injected s.c. into the hind flank with 400 μg MCA as described ( 27 ). Mice were treated early on days 0 (the day of MCA inoculation), 14, and 28 i.v. with 5 × 10 5 α-GalCer–pulsed DCs (+) or vehicle-pulsed DCs (−) and days 3, 17, and 31 i.v. with 20 μg of pORF or pIL21 DNA plasmid. Alternatively, mice were treated late on days 70, 84, and 98 i.v. with 5 × 10 5 α-GalCer–pulsed DCs (+) or vehicle-pulsed DCs (−) and on days 73, 87, and 101 i.v. with 20 μg of pORF or pIL21 DNA plasmid. Palpable sarcomas (>15 mm 2 ) were recorded and tumor-free mice were monitored for 250 d.

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1364507

15967825

20042280f9

Figure 9. IL-2 and IL-12 do not mimic the activity of α -GalCer or IL-21 in combination. All groups of five BALB/c WT mice were injected with 2.5 × 10 5 Renca tumor cells. (A) Mice were treated with: (i) 5 × 10 5 α-GalCer–pulsed DCs or vehicle-pulsed DCs i.v. on day 8; (ii) IL-2 (50,000 IU) i.p. on days 6, 7, 8, 9, and 10; or (iii) IL-12 (500 ng) i.p. on days 6, 7, 8, 9, and 10. These groups of mice received 50 μg IL-21 or PBS i.p. on days 11, 12, and 13. (B) Mice were treated with 5 × 10 5 α-GalCer–pulsed DCs or vehicle-pulsed DCs i.v. on day 8. These groups of mice received 50 μg IL-21 or PBS i.p. on days 11, 12, and 13; IL-2 (50,000 IU) i.p. on days 10, 11, 12, 13, and 14; or IL-12 (500 ng) i.p. on days 10, 11, 12, 13, and 14. In both experiments (A and B), 14 d after tumor inoculation, the lungs of the mice were harvested, and tumor colonies were counted and recorded as the mean number of colonies ± SEM. Asterisks indicate the groups in which combined α-GalCer/DC/IL-21 treatment significantly reduced lung metastases compared with all other combinations (Kruskal-Wallis: *P < 0.05).

CC BY-NC-SA

no

2022-01-13 09:01:02

J Exp Med. 2005 Jun 20; 201(12):1973-1985

PMC1373686

16216891

20051182f1

Figure 1. Dynamic of TCR microclusters during formation of a mature IS. (A) A series of images of Alexa546-H57 Fab–labeled AND TCR Tg T cell blasts interacting with planar bilayer containing ICAM-1 and agonist MHCp at times indicated. Time is relative to first detected contact area. This sequence is representative of three experiments with at least 20 cells observed in each. (B) Alexa546-H57 Fab and Fluo-LOJO dye–labeled AND TCR Tg T cell blasts interacting with planar bilayers containing ICAM-1 and agonist MHCp at early times. TIRFM of Alexa546 anti-TCR to visualize microcluster formation (top row) and wide field image of Fluo-LOJO fluorescence 4 μm above the contact plane indicates relative cytoplasmic Ca 2+ (bottom row). The TIRFM image was acquired 1 s before the Fluo-LOJO image. Representative of three experiments.

CC BY-NC-SA

no

2022-01-13 09:01:08

J Exp Med. 2005 Oct 17; 202(8):1031-1036

PMC1373686

16216891

20051182f2ac

Figure 2. Relationship of pLck 394 , pZAP-70 319 , and pLAT 191 to TCR microclusters during IS formation. Alexa546-H57 Fab–labeled AND TCR Tg T cells were incubated on bilayers containing ICAM-1 and MHCp. At different times after cell injection, the cells were fixed to yield contacts that formed between 0–0.5 min, 1–2 min, or 4–5 min. Cell were permeabilized and stained with antibodies to pLck 394 (A), pZAP 319 (B), and pLAT 191 (C) followed by Alexa488-conjugated secondary antibodies. TCR- (red in overlay) and phosphoprotein-specific antibodies (green in overlay) were imaged by wide-field fluorescence microscopy. (D) Colocalization was quantified based on the Improvision colocalization algorithm (classifier threshold 1.5×). Microclusters from at least 10 contact areas were included for each data point.

CC BY-NC-SA

no

2022-01-13 09:01:08

J Exp Med. 2005 Oct 17; 202(8):1031-1036

PMC1373686

16216891

20051182f3

Figure 3. Colocalization of pLck 394 and pZAP 319 with peripheral TCR microclusters in the IS. Cells were prepared exactly as for 4–5-min time point in Fig. 2 , and then TCR (red in overlay) and the indicated phosphoprotein (green in overlay) were imaged by TIRFM for cells stained with pLck 394 (A) and pZAP 319 (B). Examples of locations where peripheral TCR clusters colocalize with active kinases are indicated with arrowheads; positions where the large central TCR clusters contained active kinases are indicted by an arrow. The dividing line between central and peripheral structures used for calculation of the signaling efficiency is indicated in the overlay by the dotted white line. Representative of three experiments.

CC BY-NC-SA

no

2022-01-13 09:01:08

J Exp Med. 2005 Oct 17; 202(8):1031-1036

PMC1373686

16216891

20051182f4

Figure 4. Effect of inhibition of Src family kinases and actin polymerization on TCR microclusters. Alexa546-H57 Fab–labeled AND TCR Tg T cells were incubated on bilayers containing ICAM-1 and MHCp after treatment by relevant drug vehicles, 10 μM PP2 or 1 μM latrunculin A. These concentrations inhibit Src family kinases and actin polymerization, respectively, >90% in intact cells. In each case, cells were treated with the drugs for 1 h before introduction to the planar bilayers. (A) TIRFM time course of TCR clustering (top) in the presence of inhibitory concentration of PP2. Activity of PP2 was verified by lack of robust Ca 2+ elevation (bottom). (B) Reduction of pLck 394 in TCR microclusters in the presence of 100 μM PP2 versus DMSO vehicle at 5 min. (wide-field images) Similar results were obtained by TIRFM. (C) Wide-field image of unlabeled AND T cell blasts on bilayers containing Oregon Green I-E k (green in overlay) and Cy5 ICAM-1 (red in overlay) after cell treatment with vehicle, 10 μM PP2 or 1 μM latrunculin A, for 1 h before exposure to the bilayer for 1 h. The IRM image shows the contact area in darker gray. Individual fluorescence images are as indicated. The percent adhesion is indicted in the column adjacent to the images. Representative of three experiments each.

CC BY-NC-SA

no

2022-01-13 09:01:08

J Exp Med. 2005 Oct 17; 202(8):1031-1036

PMC1378109

16129702

20042469f1

Figure 1. CD68-positive cells are more abundant in TSC skin tumors than in normal-appearing skin from a patient with TSC. (A) In sections of normal-appearing skin, cells staining for CD68 (brown) are sparse in the dermis. Bar, 65 μm. (B) In sections of a periungual fibroma, there are dilated vessels and fibrosis and a more cellular stroma. Many stromal cells stain positive for CD68. Bar, 65 μm. (C) CD68-positive cells are located near vessels among CD68-negative, fibroblast-like cells. Bar, 15 μm. Similar results were observed in four angiofibromas and four periungual fibromas from six patients.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f2

Figure 2. Cells cultured from TSC skin tumors express the fibroblast marker HSP47 but not CD68. The cytoplasm of TSC fibroblasts (A) and angiofibroma cells (B) stain positive for HSP47 (green), whereas U937 cells (C), a human monocytic cell line, are negative. In contrast, the cytoplasm of TSC fibroblasts (D) and angiofibroma cells (E) are negative for CD68, and the cytoplasm of U937 cells (F) stains positive (green). Nuclei fluoresce blue with DAPI. Bar, 15 μm. Similar results were observed in cells from two other patients.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f3

Figure 3. Measurement of cytokines in culture supernatants reveals increased production of MCP-1 by angiofibroma cells. Fibroblasts from normal-appearing skin (NL) or angiofibroma cells (AF) from seven patients were incubated in 1% FBS/DMEM for 24 h, and supernatants were collected for cytokine measurement using ELISA. Lines connect paired samples for each patient. The inset shows the data as a box plot. *, P = 0.018.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f4

Figure 4. MCP-1 production is stimulated by FBS. Paired cultures of angiofibroma cells (AF) and TSC fibroblasts (NL) from two patients were seeded at 10,000 cells/well in 96-well plates, in DMEM containing 0%, 1%, 2%, or 10% FBS (1% BSA was added to the 0% FBS condition). After a 12-h incubation, MCP-1 concentration in the medium was measured by ELISA (pg/ml), and expressed per total cellular ATP (luminescence units) as an indication of cell number. Results are means ± SD of values from triplicate wells. Fresh medium with 10% FBS does not contain detectable MCP-1.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f5

Figure 5. MCP-1 produced by TSC tumor cells is chemotactic for monocytes. Conditioned medium (CM), from TSC fibroblasts (closed bars) or angiofibroma cells (open bars), CM plus control antibody, or CM plus anti-MCP-1 antibody was added to the bottom chamber of cell migration plates. THP-1 cells (a human monocytic cell line) were added to the top chamber. Cells that migrated through 8-μm pores to the feeder tray after a 2-h incubation were lysed and detected by CyQuant GR dye that exhibits enhanced fluorescence upon binding cellular nucleic acids. Results are geometric means ± SD of values from three separate migration chambers. *, P = 0.019 and **, P = 0.007 as compared with CM from TSC fibroblasts. Conditioned medium from angiofibroma cells or TSC fibroblasts contained 1640 and 60 pg/ml MCP-1, respectively. Similar results were observed in three separate experiments.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f6

Figure 6. EEF Tsc2 − / − fibroblasts produced more MCP-1 than normal (EEF Tsc2 + / + ) fibroblasts. Culture supernatants were collected after incubating EEF cells for the indicated time, and MCP-1 in the medium was measured by ELISA. Results are means ± SD of triplicate supernatants.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f7

Figure 7. Transfection of WT but not mutant TSC2 into EEF Tsc2 − / − cells inhibits MCP-1 production. EEF Tsc2 −/− cells were transfected with the indicated amounts of human TSC2 constructs and/or empty vector. In the last lane, EEF Tsc2 +/+ cells were transfected with empty vector. After transfection, cells were maintained in DMEM with 10% FBS overnight before the culture medium was replaced with DMEM containing 2% FBS; then incubation continued for an additional 24 h. The cells were harvested, and tuberin expression was detected by Western blot. Culture supernatants were collected, and MCP-1 release into the medium was measured by ELISA. Results are the mean ± SD of three separate wells. M1, mutation G294E; M2, mutation I365del; P1, polymorphism M286V; P2, polymorphism R367Q. *P, < 0.01 compared with empty vector control. Similar results were obtained in three separate experiments.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1378109

16129702

20042469f8

Figure 8. MCP-1 production by EEF Tsc2 − / − cells is inhibited by rapamycin, FTI-277, and LY294002. Rat MCP-1 was measured in culture supernatants by ELISA after incubating equal numbers of cells 24 h in serum-free medium without or with inhibitor at the indicated concentration. MCP-1 concentration was expressed per total secreted protein at the end of the incubation and is reported as the mean ± SD of triplicate wells. *, P < 0.01 compared with control. Similar results were obtained in three separate experiments.

CC BY-NC-SA

no

2022-01-13 09:01:06

J Exp Med. 2005 Sep 5; 202(5):617-624

PMC1382195

15767295

200409227f1

F igure 1. Effects of PKA activators and inhibitors on ΔR-CFTR. (A) A cell-attached recording showing that addition of 10 μM forskolin (Fsk) + 100 μM CPT-cAMP failed to increase the basal current of ΔR-CFTR. However, addition of 20 μM genistein (Gen) could potentiate the current ( n = 4). (B and C) Effects of PKI, a peptide inhibitor of PKA, on whole-cell currents from either WT- or ΔR-CFTR. For WT-CFTR, in the presence of forskolin, a very brief outward current was obtained immediately after the whole-cell configuration was formed (arrow). Then, the currents decayed rapidly. Subsequent application of genistein did not potentiate the currents. For ΔR-CFTR, basal currents were seen without any cAMP stimulant after the whole-cell configurations are formed (arrow). Subsequent application of forskolin did not alter the currents. But, genistein could potentiate the channel activity. Similar results were obtained from seven cells. Ramp I–V curves (right) were taken as marked in the raw current traces.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f2

F igure 2. Effect of PKC on ΔR-CFTR. (A) A representative whole-cell ΔR-CFTR current trace showing lack of effects of BIM, a PKC inhibitor, on the basal current. The inset shows the I–V relationships at different conditions as marked. Similar results were obtained from seven cells. (B) A continuous ΔR-CFTR current trace in a cell-attached mode. PMA, a PKC activator, did not alter the ΔR-CFTR currents. Summary of the mean currents in the presence or absence of PMA. Values were normalized by mean currents with genistein ( n = 6).

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f3

F igure 3. PKA did not alter the ATP-induced ΔR-CFTR chloride channel currents. (A) Single-channel currents were induced by ATP application 2 min after patch excision. Subsequent addition of PKA did not alter the channel activity. (B) Expanded traces of the recording in A. (C) Summary of the measured Po with 2.75 mM ATP alone and with both 2.75 mM ATP and PKA (25 U/ml) ( n = 6).

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f4

F igure 4. Effect of different concentrations of ATP on single-channel kinetics. (A) Representative single-channel ΔR-CFTR traces with different concentrations of ATP in excised inside-out patches. Each trace represents a 60-s recording. ATP concentration dependence of the mean open time (B) and the mean closed time (C). All values are represented by mean ± SEM. Overlaid open circles (○) represent the corresponding values measured for WT-CFTR channels (from Zeltwanger et al., 1999 ).

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f5

F igure 5. Mode shifts of ΔR-CFTR. (A) A single-channel recording of the ΔR-CFTR channel in an excised inside-out patch. The slow gating mode, i.e., long openings and closings of the channel, is often observed immediately after excision of the membrane patch. The slow gating mode usually switches to the fast gating mode spontaneously within a few minutes after patch excision. (B) A sample trace showing a spontaneous mode switch from the fast gating mode to the high Po mode. The mode switch occurred 4 min after the excision of membrane, and lasts for >6 min. (C) Summary of Po of ΔR-CFTR in different modes. *, P < 0.01.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f6

F igure 6. Inhibition of ATP-induced ΔR-CFTR currents by ADP. (A) Inhibitory effects of ADP on the ΔR-CFTR currents induced with different ATP concentrations. (B) The dose–response relationship between [ADP] and the magnitude of inhibition in the presence of four different ATP concentrations: (□) 75 μM ATP, (▪) 200 μM ATP, (○) 500 μM ATP, and (•) 1 mM ATP. All data points are presented as mean ± SEM of at least four values obtained from different patches. Data are fitted with the Michaelis-Menten equation.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f7

F igure 7. Effect of ADP on ΔR-CFTR single-channel current. (A) A representative single-channel current trace in an excised inside-out patch in the presence or absence of ADP. (B) Expanded traces with ATP alone (top) and with both ATP and ADP (bottom). (C) Summary of single-channel kinetic parameters ( n = 11). *, P < 0.05.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f8

F igure 8. Single-channel dwell time histograms. Events from several single-channel recordings were pulled together to obtain these dwell time histograms to determine the open and closed times distributions in the presence of 1 mM ATP, 1 mM ATP + 1 mM ADP, and 1 mM ATP + 2 mM ADP. (A) The closed time distributions can be fitted with a double exponential function in the presence of ADP, indicating the presence of a new, longer closed time constant. The data could be fitted with a single exponential (τ = ∼2 s), but in the case of 2 mM ADP, the fitted curve fails to capture nearly all the closed events >10 s (not depicted). (B) The open time dwell time histograms show that the mean open time decreases as the concentration of ADP increases.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227f9

F igure 9. Effect of ADP on the mean open time in the slow gating mode. (A) A representative single-channel ΔR-CFTR current trace in slow gating mode. (B) Expanded traces with ATP alone and with both ATP and ADP from the recording shown in A. (C) Reversible shortening of the mean open time by ADP from six patches where ΔR-CFTR channels are in slow gating mode.

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227s2

(SCHEME 2)

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382195

15767295

200409227s1

(SCHEME 1)

CC BY-NC-SA

no

2022-01-13 09:52:21

J Gen Physiol. 2005 Apr; 125(4):361-375

PMC1382337

16365163

200404104f1

Figure 1. Pik1 localizes to cytoplasmic puncta enriched in a diagnostic Golgi marker. (A) Strain BY4743 carrying pTS8 was grown to mid-exponential phase at 30°C and examined by fluorescence microscopy (left) or Nomarski (DIC) optics (right). (B) Strain YTS114 expressing Sec7-DsRed (from the TRP1 locus) and GFP-Pik1 from pTS8 were grown to mid-exponential phase, immobilized in soft agar, and examined by fluorescence microscopy using band-pass filters optimized for the detection of GFP (top left) or DsRed (top right), and by Nomarski optics (bottom right). Overlay of the GFP and DsRed images is also shown (merge).

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f2

Figure 2. Pik1 is also a resident in the nucleus. (A) Two independent fields of the same culture that was described in Fig. 1 A. Some cells (typically those with the highest total fluorescence) exhibit a strong nuclear signal (arrows). (B) Strain BY4743 carrying pTS9 were induced with galactose for 3 h, counterstained for DNA with Hoechst 33258, and viewed under the fluorescence microscope with appropriate filters to visualize GFP (left) and the DNA dye (right).

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f3

Figure 3. Nuclear import of Pik1 requires importin-β, but not importin-α. (A) An importin-α ( kap60 ts ) mutant (YTS0012 srp1-31 ts ) and its otherwise isogenic KAP60 + parent (W303-1a) carrying pTS7 were grown in SCRaf-Trp at 26°C to A 600 nm ≈ 0.7, and a portion of each culture was shifted to 37°C. After 1 h, expression of GFP-Pik1 was induced by addition of galactose, and the cells were incubated for an additional 2 h at the indicated temperature before being inspected by fluorescence microscopy. (B) The identical experiment described in A was performed with an importin-β ( kap95 ts ) mutant (SWY1313 rsl1 ts ) and its otherwise isogenic parent strain (SWY1312) carrying pTS7, except that, after galactose induction, the cells were incubated for 3 h at the indicated temperature before inspection.

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f4

Figure 4. Tethering of Pik1 at the Golgi requires Frq1. (A) Transformants of strain BY4743 carrying pTS9 and pTS10 to express either GFP-Pik1 (left) or GFP-Pik1(Δ152-191) (right), respectively, were induced with galactose for a brief period followed by shift to glucose to repress further expression, before viewing by fluorescence microscopy. (B) A frq1 Δ null mutant (YKBH9) is shown harboring both YEp352 GAL-PIK1 and pTS7, grown on galactose medium and viewed by fluorescence microscopy.

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f5

Figure 5. Frq1-GFP localizes to Golgi puncta in the cytosol. Diploid strain YTS153, expressing FRQ1-GFP from its native promoter and integrated at its endogenous locus and SEC7-DsRed from the TPI1 promoter and integrated at the TRP1 locus, was grown and viewed by fluorescence microscopy, as in Fig. 1 .

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f6

Figure 6. Pik1 is exported from the nucleus in an Msn5-dependent manner. Strains carrying conditional or null mutations in the exportin genes indicated (see Table II), and their otherwise isogenic parental strains, were transformed with either pTS6 or pTS8, as necessary, and inspected by fluorescence microscopy. Pronounced nuclear accumulation occurred only in the msn5 Δ mutant, but cytosolic puncta were also present (arrows).

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f7

Figure 7. GFP-Pik1-CCAAX is restrained in the cytosol. A wild-type ( MSN5 + /MSN5 + ) diploid (BY4743) (A) and a homozygous msn5 Δ /msn5 Δ derivative (YTS002) (B) were transformed with pTS9 (GFP-Pik1) or pTS11 (GFP-Pik1-CCAAX), as indicated, grown to mid-exponential phase, induced with galactose for 45 min, returned to glucose medium to repress further expression, and were viewed in the fluorescence microscope (left) or under Nomarski optics (right) after 1 h.

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f8

Figure 8. Pik1( Δ 10-192) accumulates in the nucleus. Diploid strain (BYB67) expressing from pRS314 GAL -myc PIK1 , pTS2 and pTS3, respectively, mycPik1 (top), mycPik1(Δ10-192) (middle) or mycPik1-CCAAX (bottom) were fixed and examined by indirect immunofluorescence using αMyc mAb 9E10 (left), after counterstaining with DAPI to reveal the nucleus (right).

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f9

Figure 9. Neither Pik1-CCAAX nor Pik1( Δ 10-192) can rescue the inviability of pik1 Δ cells. Heterozygous pik1 Δ ::KanMX4/PIK1 diploid strain (YTS68) was transformed with pTS3, pTS2 and pRS314 GAL-mycPIK1 expressing, respectively, Myc-tagged Pik1-CCAAX (A), mycPik1(Δ10-192) (B), or mycPik1 (C). Each of the transformants was induced to undergo meiosis and sporulation, and the resulting tetrads were dissected and germinated on galactose medium. Spore clones (A–D) from 10 representative tetrads (1–10) of each strain are shown.

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1382337

16365163

200404104f10

Figure 10. Interallelic complementation of the inviability of pik1 Δ cells by Pik1-CCAAX and Pik1( Δ 10-192). (A) Heterozygous pik1 Δ ::KanMX4/PIK1 diploid (YTS68) cotransformed with either pTS2 and pTS4 expressing mycPik1(Δ10-192) and mycPik1-CCAAX (left) or pTS3 and pTS4 both expressing mycPik1-CCAAX (right) was sporulated and the resulting tetrads were dissected and germinated on galactose medium. Spore clones (A–D) from 10 representative tetrads (1–10) are shown. (B) Two representative G418-resistant colonies of opposite mating type were picked, propagated on galactose (Gal) or then shifted to glucose medium (Glc), and samples of the cultures were lysed and analyzed by SDS-PAGE and immunoblotting with αMyc mAb 9E10. As markers, samples of lysates from cells expressing only mycPik1(Δ10-192) (lane 5) or mycPik1-CCAAX (lane 6) were examined. (C) Tetrad analysis, as in A, was conducted with YTS68 coexpressing either mycPik1-CCAAX and mycPik1(Δ10-192 D918A) (left) or mycPik1(D918A)-CCAAX and mycPik1(Δ10-192) (right).

CC BY-NC-SA

no

2022-01-13 07:26:13

J Cell Biol. 2005 Dec 19; 171(6):967-979

PMC1389615

14981138

200308925f1

F igure 1. State-independent block of CNGA1 and CNGA2 channels observed at 0 mV. (A and B) Current traces obtained when holding the patch at 0 mV with 20-ms steps to +20 mV at 5-s intervals. (A) CNGA1 channels in the absence of dequalinium and in the presence of 250 nM dequalinium with 2 mM (left) and 32 μM cGMP (right). Dequalinium blocked the current ∼50% under both conditions. (B) CNGA2 channels in the absence of dequalinium and in the presence of 2 μM dequalinium with 2 mM (left) and 1.8 μM cGMP (right). Dequalinium blocked the current ∼50% under both conditions. (C) Dose–response for block of CNGA1 channels (squares) and CNGA2 channels (circles) by dequalinium. Filled squares represent block in the presence of 2 mM cGMP and open squares represent block in the presence of 32 μM cGMP for CNGA1 channels. For CNGA2 channels, filled circles represent block in the presence of 2 mM cGMP and open circles represent block in the presence of 1.8 μM cGMP. The smooth curves are fits with Eq. 1 (see materials and methods ) with IC 50 = 189 nM and 192 nM for CNGA1 (high and low cGMP, respectively) and 2.4 μM (both for high and low cGMP) and the Hill coefficient = 1.4 in all cases. Error bars represent SEM, n = 5–7 patches.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925sc1

SCHEME I

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f2

F igure 2. Time course of block of CNGA1 channels during depolarizing and hyperpolarizing voltage steps. (A) Block by 50 nM dequalinium during a voltage step to +60 mV. The gray curve is a fit with a single exponential, with a time constant τ = 2.9 s. (B) Block by 50 nM dequalinium during a voltage step to −60 mV. The gray curve is a fit with a single exponential, with a time constant τ = 17 s. (C) Time course of block obtained by using a protocol in which the holding potential was held at 0 mV and pulsed for 20 ms to +20 mV every 5 s. The gray line is a fit with a single exponential, yielding τ = 59 min.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f3

F igure 3. Voltage dependence of block in CNGA1 and CNGA2 channels. (A) Block of CNGA1 channels by 50 nM dequalinium in the presence of 2 mM cGMP (filled circles) or 32 μM cGMP (open circles). The curves represent fits with Scheme I , with K Do = 4 μM and K Dc = 1.3 μM. The value of zδ was 1 in both cases. (B) Block of CNGA2 channels by 500 nM dequalinium in the presence of 2 mM cGMP (filled circles) or 1.8 μM cGMP (open circles). The curves represent fits with Scheme I , with K Do = 20 μM, and K Dc = 3.6 μM The value of zδ was 1 in both cases. Dequalinium concentrations below the IC 50 of block were used since otherwise block at depolarized potentials becomes rapidly saturated and the voltage-dependence of block cannot be correctly determined.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f4

F igure 4. State-dependent block of the S4/S5-linker chimeras by dequalinium at 0 mV. Block by different dequalinium concentrations of (A) S4/S5-CNGA1 and (B) S4/S5-CNGA2 channels. Filled symbols represent block in the presence of 2 mM cGMP and open symbols represent block in the presence of subsaturating cGMP concentrations (32 μM for S4/S5-CNGA1 and 1.8 μM for S4/S5-CNGA2). Fits to the Hill equation yielded mean IC 50 values for S4/S5-CNGA1 channels of 360 nM at 2 mM cGMP and 156 nM with 32 μM cGMP, and for S4/S5-CNGA2 channels of 2.7 μM at 2 mM cGMP and 650 nM at 1.8 μM cGMP. (C) Block of wild-type and mutant channels. A CNGA1 channel background (depicted in black) was used to construct chimeras where specific regions where replaced by the corresponding regions of CNGA2 (depicted in gray). The IC 50 values for each of the constructs obtained in the presence of saturating cGMP (2 mM, white boxes) and subsaturating cGMP (32 μM or 1.8 μM, gray boxes) are shown for each construct. For the box and whisker plot the line represent the median of the data, the box surrounds the 25th through 75th percentile, and the whiskers extend to the 5th and 95th percentiles.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f5

F igure 5. cGMP activation curves for S4/S5-chimeras. (A and C) Activation of the S4/S5-CNGA1 and S4/S5-CNGA2 chimeras by cGMP. Data were fit with Eq. 1 (see materials and methods ). The dashed lines represents the activation curve for wild-type CNGA1 (A) and CNGA2 channels (C). The K 1/2 for activation by cGMP was 66 μM for S4/S5-CNGA1 and 2.6 μM for S4/S5-CNGA2. (B and D) Fractional activation by cAMP (thin traces) was of ∼6% of the maximal current activated by cGMP (thick traces) for both wild-type CNGA1 (B, left) and S4/S5-CNGA1 channels (B, right), and 86% for wild-type CNGA2 (D, left) and S4/S5-CNGA1 (D, right)

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f6

F igure 6. Voltage dependence of block in S4/S5-CNGA1 and S4/S5-CNGA2 channels. (A) Block of S4/S5-CNGA1 channels by 50 nM dequalinium in the presence of 2 mM cGMP (filled circles) and 32 μM cGMP (open circles). The curves represent fits with Scheme I , with K Do = 4 μM and K Dc = 250 nM. (B) Block of S4/S5-CNGA2 channels by 500 nM dequalinium in the presence of 2 mM cGMP (filled circles) and 1.8 μM cGMP (open circles). The curves represent fits with Scheme I , with K Do = 14 μM and K Dc = 300 nM.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f7

F igure 7. Summary of the effects of mutations in the S4/S5 region on affinity of open and closed channels for dequalinium. Regions of sequence from CNGA1 are shown in black and regions of sequence from CNGA2 are shown in gray.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1389615

14981138

200308925f8

F igure 8. Localization of amino acids of the S4/S5 chimeras in the channel structure. (A) Sequence alignment of four types of ion channels, CNGA1, CNGA2, KcsA, and KirBac 1.1. Asterisks denote amino acids mutated in the S4-S5 linker chimeras. (B) Structure of the KcsA K + channel ( Doyle et al., 1998 ). The circle with the “I” represents the position of one of the amino acids we mutated in the S4/S5 chimeras of CNG channels. The dotted line and the question mark indicated that the region of sequence containing the other S4-S5 linker mutations is not resolved. (C) Structure of the KirBac 1.1. K + channel ( Kuo et al., 2003 ). The positions analogous to the four mutations of the S4/S5 chimeras we produced are shown as circles.

CC BY-NC-SA

no

2022-01-13 09:51:29

J Gen Physiol. 2004 Mar; 123(3):295-304

PMC1401226

15314063

200404015f1

Figure 1. Dynein/dynactin inhibition increases the length of spindle microtubules in the presence or absence of centrosomes. (A–C) Tubulin distribution in untreated spindles during live recordings. (D–G) p150-CC1 addition (2 μM, ∼3 min before image at t = 0) caused spindles to increase in length. (H and I) Higher magnified spindle pole regions indicated in F (Videos 1 and 2). (J) p150-CC1 was added to assembled spindles, samples were fixed after 8 or 15 min, spindle lengths were measured (mean ± SD, n = 15, two independent experiments), and normalized to the length of untreated spindles (40 μm). (K–M) Spindles fixed 8 min after addition of control buffer (K), 2 μM p150-CC1 (L), or 1 mg/ml 70.1 (M) (tubulin, red; DNA, blue). (N–P) Higher magnified, contrast-adjusted regions indicated in K–M, respectively. (Q and R) Spindles assembled in 18 μm p50 dynamitin were treated with control buffer (Q) or 2 μm p150-CC1 (R) and fixed after 15 min (tubulin, red; DNA, blue). (S and T) Spindles assembled in the absence of centrosomes, around DNA-beads (tubulin, red; DNA, blue). (S) Buffer control. (T) p150-CC1–treated (2 μM, 8 min). (U and V) Higher magnified, contrast-adjusted regions indicated in R. Times are in min:s. Bars, 10 μm.

CC BY-NC-SA

no

2022-01-13 07:23:08

J Cell Biol. 2004 Aug 16; 166(4):465-471

PMC1401226

15314063

200404015f2

Figure 2. Dynactin inhibition with p150-CC1 suppresses microtubule depolymerization at spindle poles. Spindle microtubule dynamics were analyzed using fluorescent speckle microscopy. (A) Tubulin speckles in a control spindle. (B) Polewards velocities of tubulin speckles in control (white bars; 2.1 ± 0.3 μm/min, mean ± SD), or p150-CC1–treated (2 μM p150-CC1, black bars; 2.1± 0.2 μm/min, mean ± SD) spindles ( n = 12 for each condition, 120 speckles). Velocities were binned in 0.5 μm/min increments. (C–E) Images from a time-lapse video of a p150-CC1–treated spindle (2 μM p150-CC1 added ∼3 min before image at t = 0) showing tubulin speckles (C and D) and tubulin distribution (E). The black lines in A, C, and D indicate the regions used to generate the kymographs shown in F and G, respectively (Videos 3 and 4). Times are in min:s. Bars, 10 μm.

CC BY-NC-SA

no

2022-01-13 07:23:08

J Cell Biol. 2004 Aug 16; 166(4):465-471

PMC1401226

15314063

200404015f3

Figure 3. NuMA inhibition with LGN-N increases the length of spindle microtubules, in the presence or absence of centrosomes. (A–D) Tubulin distribution in an LGN-N–treated spindle (0.7 μM, added ∼3 min before image at t = 0) during live recordings (Video 5). (E) LGN-N was added to assembled spindles, samples were fixed after 8 or 15 min, spindle lengths were measured (mean ± SD, n = 15, two independent experiments), and normalized to the length of untreated spindles (40 μm). (F–H) Spindles assembled in the absence of centrosomes, around DNA beads (tubulin, red; DNA, blue). (F) Buffer control. (G) LGN-N–treated (2 μM, 8 min). (H) Higher magnified image of the region indicated in G. Times are in min:s. Bars, 10 μm.

CC BY-NC-SA

no

2022-01-13 07:23:08

J Cell Biol. 2004 Aug 16; 166(4):465-471

PMC1401226

15314063

200404015f4

Figure 4. Kif2a is required for bipolar spindle assembly and the regulation of spindle microtubule length. (A) Western blot of Xenopus egg extracts stained with anti-Kif2a. Molecular weight standards are shown. (B–E) Anti-Kif2a inhibits bipolar spindle assembly. Anti-Kif2a (0.7 mg/ml; B and C) or control buffer (D and E) were added at the start of assembly reactions. (B and D) Tubulin alone. (C and E) Overlay (tubulin, red; DNA, blue). (F–K) Anti-Kif2a (0.7 mg/ml) was added to assembled spindles. (F and G) 8 min after antibody addition. Long microtubule bundles extended beyond (white arrowheads), and buckled (green arrowheads) within the spindle. (F) Tubulin alone. (G) Overlay (tubulin, red; DNA, blue). (H–K) Real-time analysis of a spindle treated with anti-Kif2a (added ∼3 min before image at t = 0; Video 6). Times are in min:s. Bars, 10 μm.

CC BY-NC-SA

no

2022-01-13 07:23:08

J Cell Biol. 2004 Aug 16; 166(4):465-471

PMC1401226

15314063

200404015f5

Figure 5. Treatment with p150-CC1 or 70.1 displaces Kif2a, but not MCAK from spindle poles. Assembled spindles, after addition of buffer, p150-CC1, or 70.1, were processed for immunofluorescence. MCAK staining in control (A and D), p150-CC1–treated (2 μM, 15 min; B and E), and 70.1-treated (1 mg/ml, 15 min; C and F) spindles. (A–C) Overlays (tubulin, red; DNA, blue; MCAK, green). (D–F) MCAK alone. (G–I) Line scans of fluorescence intensity (MCAK, green; tubulin, red; arbitrary units) across the pole to pole axis of the spindles shown in A–C. Kif2a staining in untreated (J and M), p150-CC1–treated (2 μM, 15 min; K and N), and 70.1-treated (1 mg/ml, 15 min; L and O) spindles. (J–L) Overlays (tubulin, red; DNA, blue; Kif2a, green). (M–O) Kif2a alone. (P–R) Line scans of fluorescence intensity (Kif2a, green; tubulin, red; arbitrary units) across the pole to pole axis of the spindles shown in (J–L). (S and T) Spindles were treated for 15 min with p150-CC1 (4 μM) or control buffer and the relative amount of Kif2a (S), or MCAK (T), and tubulin associated with partially purified spindle pellets was analyzed by immunoblotting. (U) Quantitation of spindle-associated Kif2a and MCAK relative to tubulin from measurements of immunoblot band intensities (mean ± SD, two independent experiments), normalized to intensities from untreated spindles. Bars, 10 μm.

CC BY-NC-SA

no

2022-01-13 07:23:08

J Cell Biol. 2004 Aug 16; 166(4):465-471

PMC1414780

15623895

200409197f1

F igure 1. The ILT mutant channel. (A) The S4 sequence for Shaker and for the ILT mutant channel. Charged residues are denoted in bold and numbered. (B) Cartoon illustrating activation and opening in WT and ILT channels. Only two subunits of the channel are shown, with the positively charged helix S4 and the S6 helix. The long gray arrow represents a voltage axis ranging from −150 to 200 mV. In the WT channel (top), activation and opening are tightly coupled and the activated state is short-lived (denoted by a bracket around the activated state). In the ILT channel (bottom), activation is negatively shifted, whereas opening is positively shifted. Cartoon based on results from Ledwell and Aldrich (1999) .

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f2

F igure 2. S6 gate in ILT channels is closed in the activated conformation. (A) Cartoon showing that the NH 2 -terminal ball can block the channel only when the S6 gate is open. (B) ILT+ball channels show fast inactivation in excised inside-out macropatches. Inset shows voltage protocol used, HP = −100 mV. (C) Fast inactivation in ILT+ball channels takes place over the voltage range of opening, not activation. Steady-state (S.S) prepulse fast inactivation for ILT+ball channels (closed circles) measured from peak amplitude of current during a 20-ms test pulse to +180 mV following a 100-ms voltage step to the specified potential, HP = −100 mV with a 50-ms prestep to −120 mV. G–V from ILT channels (open circles) constructed as described in materials and methods , Q–V (open squares) measured from integrated OFF gating currents of ILT W434F channels. Solid lines are Boltzmann fits to the data. Fit parameters: ILT Q–V, V 1/2 = −70.4 mV, slope factor = 16.3 mV, representative of n = 8; ILT+ball prepulse inactivation curve, V 1/2 = +77.3 mV, slope factor = 13.3, average of data from 11 patches; ILT G–V, V 1/2 = +91.8 mV, slope factor = 14.9, average of data from 8 patches. (D) Two voltage pulses to +180 mV, from a prestep to −120 mV and from an ensuing prestep to 0 mV, yield currents with similar amplitude in ILT+ball channels. Representative trace of n = 6.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f3

F igure 3. S4 attached TMRM senses protein motion over voltage range of ILT channel opening. (A) Normalized steady-state F–V for 359C WT W434F (open diamonds) and for 359C ILT W434F (closed circles). F–Vs are normalized to the amplitude of single Boltzmann fit for 359C WT and a double Boltzmann fit for 359C ILT (fits are solid lines). Fit parameters: 359C WT W434F V 1/2 = −43.8 mV, slope factor = 7.3, representative of n = 13; 359C ILT W434F V1 1/2 = −66.8 mV, slope factor = 17.2, V2 1/2 = +105.6 mV, slope factor = 24.1, representative of n = 18. Traces on top show the fluorescence intensity change evoked from a prestep of −120 mV to voltage step to 0 mV (gray) or 180 mV (black) for 359C ILT W434F channels. HP = −80 mV. (B and C) Superimposition of the Q–V from 359C-TMRM ILT W434F channels (B, filled circles), and the G–V from 359C-TMRM ILT channels (C, filled circles) with the F–V from 359C-TMRM ILT W434F channels (open circles). F–V and Q–V measured in two-electrode voltage clamp configuration, and G–V measured from excised inside-out macropatches. Each data point is a mean of n = 5 for the Q–V, n = 9 for G–V, and n = 9 for F–V. Solid lines are Boltzmann fits to the data. Fit parameters: Q–V V 1/2 = −67.3 mV, slope factor = 25.8; G–V V 1/2 = 94.3 mV, slope factor = 22; F–V double Boltzmann fit parameters: V1 1/2 = −69.5 mV, slope factor = 17.6, V2 1/2 = +105.1 mV, slope factor = 23.3.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f4

F igure 4. Opening rearrangement sensed at four sites in S4 and proximal S3–S4 linker. Steady-state fluorescence behavior of TMRM attached to sites 351C (A), 356C (B), 358C (C), and 361C (D). Each panel shows the F–Vs for TMRM attached to the specified site in the WT W434F background (open diamonds) and in the ILT W434F background (filled circles). Open circles show G–Vs measured from excised inside-out macropatches for ILT channels labeled with TMRM at the specified site. Traces on top of each graph show the fluorescence intensity change in response to a step to 0 mV (gray) and 180 mV (black) from a prestep to −120 mV in the ILT W434F background. HP = −80 mV. Different amounts of separation between the WT and ILT F–Vs at the four positions likely reflect different impacts on gating of the cysteine mutation and TMRM labeling. F–Vs are representative of n = 7 for 351C ILT, n = 6 for 351C WT, n = 3 for 356C ILT, n = 10 for 356C WT, n = 3 for 358C ILT, n = 6 for 358C WT, n = 6 for 361C ILT, n = 4 for 361C WT. Fit parameters for G–Vs: 351C ILT V 1/2 = 113.6 mV, slope factor = 21.2, n = 3; 356C ILT V 1/2 = 96.1 mV, slope factor = 20.5, n = 6; 358C ILT V 1/2 = 94.3 mV, slope factor = 22.0 mV, n = 6; 361C ILT V 1/2 = 102.9 mV, slope factor = 19.5, n = 9.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f5

F igure 5. 4-AP eliminates the fluorescence change over the voltage range of opening for 359C-TMRM ILT. (A) Fluorescence changes for 359-TMRM ILT W434F channels from the same cell in the voltage range of opening in the absence (left) and presence (right) of 2 mM 4-AP added to the bath solution. Voltage protocol is shown as inset. (B) Steady-state fluorescence plotted as a function of voltage for channels in the absence of 4-AP (open circles) and in the presence of 2mM 4-AP (filled circles).

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f6

F igure 6. Slow inactivation in ILT channels over voltage range of S4's gating motion. (A, i) Superimposed currents in response to a 1-s depolarizing step to +150 mV for ILT channels (gray trace) and ILT/T449V channels (black trace) show that the T449V mutation slows slow inactivation of ILT channels. HP = −100 mV, post-step voltage = +40 mV. (ii) Voltage protocol (left, bottom) and currents (left, top) used for measuring the voltage dependence of slow (P-type) inactivation from conducting ILT channels in excised inside-out macropatches. Interval between sweeps = 5 s at an HP of −100 mV. The fraction of current after inactivation (FCAI, right, filled symbols) was plotted from the amplitude of the test pulse following the 1-s inactivating pulses after correcting for run-down in ionic current from patch (see materials and methods ). G–V for ILT channels measured from the same patch (right, open symbols). Data are representative of n = 3 patches. Solid lines are Boltzmann fits to the data. Fit parameters for data from three patches: for ILT Slow inactivation, V 1/2 = +108.6 ± 9.8 mV; G–V, V 1/2 = +101.4 ± 6.9 mV. (B) C-type inactivation does not occur from the activated state. Representative F–Vs for 359C WT W434F (left,) and 359C ILT W434F (right), from an HP of −80 mV (open symbols) or 0 mV (closed symbols). Interval between traces = 9 s. Solid lines represent single Boltzmann fits for WT and double Boltzmann fits for ILT. For 359C WT W434F, representative of n = 3: −80 mV hold, V 1/2 = −41.4 mV, slope factor = 9.3; 0 mV hold, V 1/2 = −48.9 mV, slope factor = 8.3. For 359C ILT W434F, representative of n = 4: double Boltzmann fit with −80 mV hold, V1 1/2 = −55.9 mV, slope factor 1 = 14.0, V2 1/2 = +111.4 mV, slope factor 2 = 20.5; 0 mV hold, V1 1/2 = −58.6 mV, slope factor 1 = 14.1, V2 1/2 = +111.1 mV, slope factor 2 = 23. (C) ILT channels undergo C-type inactivation. (i) Tail currents following a voltage step to 0 mV for 449V WT (left) and 470C/449V WT (right) channels. Voltage protocol is shown as inset; t a is the duration of voltage step = 10, 40, 100, 400, 1,000, or 1,400 ms. (ii) Tail currents for 449V ILT and 470C/449V ILT channels following a voltage step to 150 mV with t a = 20, 100, 200, 400, 1,000, or 1,500 ms. Recordings are from excised inside-out macropatches in symmetric (140 mM K + ) conditions. Tail current voltage for ILT channels is +40 mV since the inward tails at −80 mV are fast and hard to distinguish from the capacitive transient.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f7

F igure 7. S4s move cooperatively over the voltage range that drives gating. (A) Subunit stoichiometry for hetero-tetrameric channels and their controls. * indicates a labeled subunit. Symbols shown under cartoons are same as used in graphs B and C. (B) Mean of F–Vs from labeled ILT*(WT) 3 (filled circles), (WT*) 4 (open, inverted triangles), and (ILT * ) 4 channels (open circles). Solid lines are Boltzmann fits to the data. Fit parameters: ILT*(WT) 3 V 1/2 = −50.9 mV, slope factor = 19, n = 6; (WT*) 4 V 1/2 = −39.1 mV, slope factor = 8.2, n = 4. For (ILT*) 4 , double Boltzmann fit parameters: V1 1/2 = −69.5 mV, slope factor = 17.6, V2 1/2 = +105.1 mV, slope factor = 23.3, n = 11. (C) Mean of F–Vs from labeled WT*(ILT) 3 (filled, inverted triangles). Fit parameters for WT*(ILT) 3 double Boltzmann fit V1 1/2 = −35.7 mV, slope factor = 12.0, V2 1/2 = 28.3 mV, slope factor = 20.5, n = 11; fits parameters to (WT*) 4 and (ILT * ) 4 channels are same as in B.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1414780

15623895

200409197f8

F igure 8. A model for channel opening. (A) Mapping of Shaker ILT positions onto the crystal structure of KvAP's isolated voltage sensing (S1–S4) domain ( Jiang et al., 2003a ) shows ILT residues to face away from other membrane segments in direction deduced to interact with the pore domain in the activated conformation ( Gandhi et al., 2003 ). ILT residues are colored. P.D., pore domain. (B) Model for association between activation and gating motions of S4 and opening of the S6 gate. Top layer shows a side view of two subunits of the channel; dotted lines point to a top view of the channel with all four subunits shown. The S4s (orange cylinders) move independently (or with a mild cooperativity) of each other during activation, and do not exert force on the S6 gate (green cylinders). In the activated state, each S4 interacts with its neighboring S5 (blue oval, top view), which is denoted by dashed line. From the activated state, the S4s undergo a cooperative motion in which each S4 places strain on the S6 gate via S4–S5 (hook and spring representation). The interaction of an S4 with its neighboring S5 is stronger in ILT, hence a strong depolarization is needed to release S4 from its activated conformation to undergo its final motion.

CC BY-NC-SA

no

2022-01-13 09:51:38

J Gen Physiol. 2005 Jan; 125(1):57-69

PMC1424221

9744873

JCB9806103.f1

Figure 1 Phosphorylation-dependent routing of furin in early endosomes ( top ). Epitope-tagged furin showing the FLAG insertion ( cross-hatched ), the catalytic ( shaded ) and transmembrane domains ( stippled ), as well as the sequence of the cd with CKII phosphorylation site substitutions. ( Bottom ) BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 10) expressing either fur/f with the native cytosolic domain ( A and B ) or with serine to aspartic acid substitutions within the CKII site (fur/f-DDD, C–E ). At 6 h postinfection, the cells were incubated with mAb M1 (15 μg/ml) and TRITC-transferrin (40 ng/ml) ( E ) for 1 h before fixation and processing for immunofluorescence. The cells in B were treated with 100 nM tautomycin during the uptake period. Internalized mAb M1 was visualized using FITC goat anti–mouse IgG ( A–D ).

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f2

Figure 2 Internalization of furin is dependent upon dynamin function. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f and either native dynamin I ( A and C ) or the dominant-negative dynamin construct K44E ( B and D ) in the presence of 10 μM hydroxyurea. At 16 h postinfection, cells were incubated with either mAb M1 ( A and B ) or TRITC-transferrin ( C and D ) for 1 h in culture before fixation and analysis by immunofluorescence. The extended culture time postinfection was required to allow sufficient expression levels for optimum K44E block.

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f3

Figure 3 Recycling of phosphorylated furin from endosomes to the cell surface. ( A ) HeLa TS-Dyn I cells were infected with vaccinia recombinants (m.o.i. = 10) expressing fur/f mutants mimicking either nonphosphorylated (fur/f ADA ) or constitutively phosphorylated furin (fur/f DDD ). After accumulation at the cell surface during incubation at nonpermissive temperature (37°C) for 6 h, surface proteins were biotinylated (refer to Materials and Methods) and then allowed to internalize for the indicated times at permissive temperature (31°C). The total ( T ) and internalized ( I ) pools of labeled furin were then determined by immunoprecipitation and Western analysis. ( B ) The percent of furin internalized at each time point was quantified by densitometric analysis (data averaged from four independent experiments, error bars = SEM). ( Inset ) Double-strip analyses were conducted in which the pool of internalized furin following an initial 20-min chase period was assessed for reexpression at the cell surface. After a second 20-min chase, total biotinylated furin was compared with surface localized furin using a second MesNa strip (− and +, respectively).

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f4

Figure 4 Endocytic sorting of furin requires the phosphorylated cd binding protein PACS-1. Fur/f was expressed in control cells ( A , B , E , and F ) and PACS-1–deficient antisense cells ( C , D , G , and H ) using vaccinia recombinants (m.o.i. = 10). At 4 h postinfection, the cells were exposed to mAb M1 as a marker for endocytosed furin and TRITC-labeled transferrin ( B and D ) for either 1 h ( A–D , F , and H ) or 10 min. ( E and G ) before fixation and processing for immunofluorescence microscopy. The cells in A–D were exposed to 100 nM tautomycin during the 1-h incubation period with mAb M1 and transferrin. Internalized mAb M1 was visualized using FITC-conjugated goat anti–mouse IgG2b ( A , C , and E–H ).

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f5

Figure 5 Dephosphorylation of furin by purified phosphatase. PP1 and PP2A catalytic subunits were incubated with either 32 P-labeled GST-Fur cd fusion protein ( GST–furin cd ) phosphorylated with CKII, or a 32 P-labeled control substrate (phosphorylase a, Phos. a ) for the indicated times (in min). Aliquots from the dephosphorylation reactions were then separated by SDS-PAGE and analyzed by autoradiography. Parallel experiments analyzed by quantitative filter paper assays also indicated no measurable dephosphorylation of the furin-cd by either enzyme (data not shown).

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f6

Figure 6 Endogenous furin-directed phosphatase activity. ( A ) Extracts from bovine brain (shown) and cultured cells were incubated with 32 P-labeled GST–Fur cd in vitro in the absence ( Control ) or presence of 100 nM tautomycin for the indicated times (in min). Aliquots of the reactions were separated by SDS-PAGE and analyzed by autoradiography. ( B ) Affinity chromatography of endogenous furin-directed phosphatase activity in BSC-40 cells. Cell extract was applied to a microcystin affinity column and the bound phosphatase eluted with 3M NaSCN. Aliquots of the original extract ( Load ), the unbound protein ( FT ), and eluted material ( E1–3 ) were assayed for total PP1/PP2A activity using 32 P-labeled phosphorylase a ( open bars ) and furin-directed phosphatase activity using 32 P-labeled GST–Fur cd ( hatched bars ). The total activities were calculated based on fraction volume and expressed as pmol/min. Assays were performed in triplicate with relative error (one standard deviation) of less than 5%. Similar results were obtained in several independent experiments.

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f7

Figure 7 Isoform-specific dephosphorylation of furin by PP2A. ( A ) Baculovirus recombinants (m.o.i. = 2) were used to express PP2A subunits, either alone or in combination, in Sf9 cells. At 64–72 h postinfection, cells were harvested and lysates were assayed for phosphatase activity with both 32 P-labeled phosphorylase a ( open bars ) and phosphorylated 32 P-labeled GST–Fur cd ( hatched bars ). Noninfected Sf9 cells (data not shown) expressed a detectable level of endogenous furin-directed phosphatase activity which was not increased by expression of the catalytic subunit either alone or in combination with the A subunit ( C , and A + C , respectively). The C and A subunits were also expressed with either the α or β isoforms of the B family of regulatory subunits ( A + C + Bα and A + C + Bβ , respectively), as well as the α subunit from the unrelated B′ family ( A + C + B′α ). As a control, lysates from A + C + Bβ– expressing cells were exposed to 100 nM tautomycin and okadaic acid (+ Inhib. ) during the phosphatase assays. Assays were performed in triplicate with relative error <5%. The data shown are representative of several independent experiments. ( B ) Western analysis of PP2A subunit expression. Lysates of Sf9 cells infected with baculovirus recombinants encoding the indicated PP2A subunits were resolved by SDS-PAGE and screened by Western blot using antisera specific for the catalytic subunit or the Bα, Bβ, or B′α regulatory subunits.

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f8

Figure 8 Disruption of endogenous PP2A affects furin trafficking in vivo. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f alone ( A and B ) or in combination with viruses expressing either SV-40 small t ( C ) or truncated and inactive small t mut3. At 6 h ( D ) postinfection, the cells were incubated with mAb M1 for 1 h in culture before fixation. The cells in B were incubated in the presence of 100 nM tautomycin during mAb M1 uptake The cells were then processed for immunofluorescence to localize internalized furin.

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424221

9744873

JCB9806103.f9

Figure 9 Model of furin trafficking in the endosomal system. Furin molecules are internalized via dynamin-sensitive, clathrin-dependent endocytosis. Within early endosomes shared by TfR, furin phosphorylated by CKII is directed toward a cell surface recycling pathway by virtue of its selective interaction with PACS-1, which links phosphorylated furin to components of the clathrin sorting machinery ( Wan et al., 1998 ). This endosome to plasma membrane recycling pathway mirrors the local cycling loop which localizes furin to the TGN ( Wan et al., 1998 ). Furin molecules dephosphorylated by PP2A isoforms containing B regulatory subunits are sorted from the plasma membrane/early endosome cycling loop to a TGN retrieval pathway. Black shading of either the tyrosine or AC motifs denotes their “active” state whereas gray shading reflects a “silencing” of these signals.

CC BY-NC-SA

no

2022-01-13 07:15:32

J Cell Biol. 1998 Sep 21; 142(6):1399-1411

PMC1424222

9412467

JCB.29208f1a

Figure 1 ( A ) Schematic of epitope-tagged furin ( Fur/f ) and COOH-terminal cytosolic domain truncated forms. The NH 2 -terminal cross-hatched box shows the FLAG epitope inserted COOH-terminal to the autoproteolytic maturation site ( Molloy et al., 1994 ). The FLAG epitope cross-reacts with both mAbs M1 and M2. Binding of the mAb M1 requires the free amino terminus of the FLAG tag, whereas mAb M2 does not. Because autoproteolytic cleavage of the furin propeptide is localized to the RER ( Molloy et al., 1994 ), all furin molecules trafficking in the TGN/endosomal compartments are capable of binding both mAbs. The lightly shaded area represents the catalytic domain with the Asp (D), His (H) and Ser (S) residues that form the catalytic triad, the hooked ovals represent NH 2 -linked carbohydrates and the dark-stippled box represents the membrane spanning domain. The sequence of the 56-amino acid cd is shown including the location of each of the COOH-terminal truncation sites. The Tyr-based and di-leucine-like internalization motifs, as well as the acidic cluster necessary for TGN localization of furin are underlined. ( B ) Two-hybrid analysis of furin cd truncations (bait), with library clone TP107, is identified in the initial screen. Yeast cells were cotransformed with pTP107 (Leu − medium) and bait plasmids (Trp − medium) expressing either furin cd or one of the furin cd truncation mutants ( A ) and selected for growth on Leu − /Trp − medium. Colonies from the Leu − /Trp − plates were streaked onto His- plates and scored for growth.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f2

Figure 2 Identification of TP107 as mouse ABP-280. ( A ) Alignment of TP107 and ABP-280. Shown are the nucleic acid sequence for the TP107 insert ( top ), the corresponding TP107 open reading frame ( middle ), and the amino acid sequence of the corresponding region of human ABP-280 ( Gorlin et al., 1990 ). Species-specific amino acid changes are shown in italics. ( B ) Schematic of ABP-280. ABP-280 is a homodimer composed of two 280-kD subunits, each of which contains an amino-terminal actin binding domain followed by 24 repeats of a structurally conserved 96-amino acid β-sheet structure ( rectangles ). Hinge regions are located between repeats 15/16 and 23/24. The centrally placed hinge region permits the orthogonal positioning of cortical microfilaments required for the “sol-gel” transition states of cytosol, as well as the formation of lamellapodia necessary for cell crawling. The COOH-terminal 24th repeat contains the ABP-280 dimerization domain. Alignment of TP107 with the reported sequence for human ABP-280 shows that it is contained within the region bridging the 13th and 14th repeats (residues 1490–1607 in human ABP-280).

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f3

Figure 3 Interaction of ABP-280 and furin. ( A ) Binding in vitro of HistagABP-280 to GST-furin cd. 6 μg of a His-tagged construct containing residues 1490–1607 of mouse ABP-280 was combined with 10 μg of GST or a GST fusion protein containing the entire furin cd ( GST-F cd ). Glutathione agarose was then added and bound proteins were removed from the washed beads with SDS-sample buffer and separated by SDS-PAGE (10% acrylamide). Proteins were detected by staining the gel with Coomassie Blue R250. The positions of molecular weight standards are shown on the right. ( B ) Coimmunoprecipitation of ABP-280 and fur/f. Replicate plates of BSC-40 cells were infected with wild-type vaccinia virus or vaccinia recombinants expressing either fur/f or the furin cd truncation mutant fur/fR 739 t depicted in Fig. 1 (moi = 5). At 16 h after infection, the cells were harvested and the clarified cell extracts were incubated overnight with the anti– filamin(ABP-280) mAb ( Gorlin et al., 1990 ). Samples were then treated with protein G Sepharose and bound proteins dissolved in SDS-sample buffer, separated by SDS-PAGE and transferred to nitrocellulose. Coimmunoprecipitating FLAG epitope-tagged furin constructs were detected by incubating the blot with mAb M1. Parallel plates of total cell extract show that equivalent amounts of mAb M1 immunoreactive fur/f constructs were expressed in each sample (data not shown). The positions of molecular weight standards are shown on the right. ( C ) Coimmunoprecipitation of ABP-280 with cell surface fur/f. Parallel plates of BSC-40 cells were infected with either wild-type vaccinia virus or the recombinant expressing fur/f. At 6 h after infection, the cells were placed on ice and cell surface molecules were biotinylated as described in Materials and Methods. The cells were then harvested and subjected to immunoprecipitation with the anti–ABP-280 mAb as described in B. The immunoprecipitates were boiled in mRIPA containing 1% SDS, diluted 10-fold in buffer, and furin molecules immunoprecipitated with the anti–furin cd antiserum. The immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and biotinylated proteins were detected by chemiluminescence ( left ). The blot was then reprobed with the anti–furin cd antiserum and developed with alkaline phosphatase ( right ). 100 kD fur/f is marked. The smaller 82 kD biotinylated furin protein (*) represents a degradation product of the endoprotease, generated under these conditions, that still contains the intact cytosolic domain.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f4

Figure 4 Quantitative internalization of [ 125 I]-mAb M1 and [ 125 I]-transferrin into M2 and A7 cells. ( A ) Parallel plates of M2 (□) and A7 (▪) cells were infected with VV: hfur/f (moi = 10). 4 h after infection, [ 125 I]-mAb M1 was added to the culture medium. Control plates were placed on ice immediately and the remaining samples were incubated at 37°C for the indicated times, and then transferred to ice. At the indicated times, the supernatants were removed, the cells washed twice, and then incubated with Proteinase K. The detached cells were suspended in fresh medium, pelleted through a serum pad, and the amount of internalized [ 125 I] was determined. For each time point, the counts from the control sample were subtracted from the value of the assay sample. Experiments using non– epitope-tagged furin showed no detectable increase in signal over background. All time points were measured in triplicate. Bars indicate standard deviations. ( B ) The measurement of [ 125 I]-transferrin uptake into M2 (○) and A7 (•) cells was performed as described in Materials and Methods. Cells were incubated with 35 nM [ 125 I]-transferrin for the indicated times at 37°C. At the end of each time point, cells were transferred to 4°C and washed with mild acid to remove cell surface transferrin. The cells were solubilized with NaOH and counted in a gamma counter. Binding assays at 4°C were performed simultaneously with each assay to determine the number of transferrin receptors on the cell surface.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f5

Figure 5 Localization and recycling of epitope-tagged furin. Parallel plates of BSC-40 ( A and B ), M2 ( C and D ), and A7 cells ( E and F ) grown on glass coverslips were infected with VV:hfur/f (moi = 10). At 4 h after infection, mAb M1 (6 μg/ml final concentration) was added to the culture media for an additional hour. The cells were then fixed and permeabilized, and the samples were incubated with mAb M2 to detect the steady state distribution of furin staining. The samples were then processed for immunofluorescence microscopy. The mAb M1 was detected using a goat anti–mouse IgG 2b -FITC secondary antibody ( A , C, and E ) and mAb M2 was visualized using a goat anti–mouse IgG 1 -TXR antibody ( B , D , and F ). The exposure time in C was adjusted to equal the exposure time in E to compare directly the intensity of fluorescence staining between samples.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733