Volume 22, Issue 5, Pages (January 2018)

Slides:



Advertisements
Similar presentations
Volume 16, Issue 6, Pages (August 2016)
Advertisements

The ER-Mitochondria Tethering Complex VAPB-PTPIP51 Regulates Autophagy
Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis 
Takashi Tanaka, Michelle A. Soriano, Michael J. Grusby  Immunity 
Kirill Kiselyov, Gregory A Mignery, Michael X Zhu, Shmuel Muallem 
Young Kwon, Thomas Hofmann, Craig Montell  Molecular Cell 
Volume 81, Issue 1, Pages (January 2014)
AMP Is a True Physiological Regulator of AMP-Activated Protein Kinase by Both Allosteric Activation and Enhancing Net Phosphorylation  Graeme J. Gowans,
A Rac-cGMP Signaling Pathway
Monica C. Rodrigo-Brenni, Erik Gutierrez, Ramanujan S. Hegde 
Volume 21, Issue 12, Pages (December 2017)
Volume 110, Issue 11, Pages (June 2016)
Feng Zhang, Jiazhong Shi, Chunjing Bian, Xiaochun Yu  Cell Reports 
Yongli Bai, Chun Yang, Kathrin Hu, Chris Elly, Yun-Cai Liu 
Volume 22, Issue 13, Pages (March 2018)
Volume 11, Issue 12, Pages (June 2015)
Volume 18, Issue 13, Pages (March 2017)
Volume 23, Issue 2, Pages (July 2006)
Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number
Robert L.S Perry, Maura H Parker, Michael A Rudnicki  Molecular Cell 
Long-Range Ca2+ Signaling from Growth Cone to Soma Mediates Reversal of Neuronal Migration Induced by Slit-2  Chen-bing Guan, Hua-tai Xu, Ming Jin, Xiao-bing.
Volume 20, Issue 4, Pages (July 2017)
Lysophospholipid Receptor-Mediated Calcium Signaling in Human Keratinocytes  Karin Lichte, Roberto Rossi, Kerstin Danneberg, Michael ter Braak, Ulrich.
Volume 137, Issue 4, Pages (October 2009)
Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling
Arachidonic acid induces ERK activation via Src SH2 domain association with the epidermal growth factor receptor  L.D. Alexander, Y. Ding, S. Alagarsamy,
Alex M. Agelidis, Satvik R. Hadigal, Dinesh Jaishankar, Deepak Shukla 
Volume 46, Issue 4, Pages (May 2005)
Volume 55, Issue 4, Pages (April 1999)
Volume 15, Issue 6, Pages (June 2012)
Volume 58, Issue 6, Pages (June 2008)
Tropomyosin Regulates Cell Migration during Skin Wound Healing
Volume 19, Issue 4, Pages (April 2017)
Meilin Wu, James E. Robinson, William J. Joiner  Current Biology 
Volume 12, Issue 9, Pages (September 2015)
Volume 13, Issue 1, Pages (January 2008)
Volume 17, Issue 9, Pages (November 2016)
Volume 18, Issue 11, Pages (March 2017)
Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period  Kyung-Seok Han, Samuel F. Cooke, Weifeng Xu  Cell.
Volume 23, Issue 2, Pages (April 2018)
Volume 57, Issue 2, Pages (January 2008)
Melissa L. Ehlers, Barbara Celona, Brian L. Black  Cell Reports 
Lysine 63 Polyubiquitination of the Nerve Growth Factor Receptor TrkA Directs Internalization and Signaling  Thangiah Geetha, Jianxiong Jiang, Marie W.
Kentaro Abe, Masatoshi Takeichi  Neuron 
GRM7 Regulates Embryonic Neurogenesis via CREB and YAP
Volume 12, Issue 1, Pages (July 2015)
Volume 21, Issue 10, Pages (December 2017)
Calcium Release from Stores Inhibits GIRK
Extracellular Regulated Kinase Phosphorylates Mitofusin 1 to Control Mitochondrial Morphology and Apoptosis  Aswin Pyakurel, Claudia Savoia, Daniel Hess,
Volume 96, Issue 6, Pages (March 1999)
ULK1 Phosphorylates and Regulates Mineralocorticoid Receptor
Dan Yu, Rongdiao Liu, Geng Yang, Qiang Zhou  Cell Reports 
Volume 22, Issue 6, Pages (February 2018)
Volume 21, Issue 1, Pages (October 2017)
Volume 129, Issue 2, Pages (April 2007)
Volume 16, Issue 1, Pages (January 2006)
Mechanism of Persistent Protein Kinase D1 Translocation and Activation
Volume 25, Issue 4, Pages e4 (October 2018)
Volume 125, Issue 4, Pages (May 2006)
Volume 83, Issue 5, Pages (November 2002)
Shrestha Ghosh, Baohua Liu, Yi Wang, Quan Hao, Zhongjun Zhou 
TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function
Volume 71, Issue 6, Pages (September 2011)
In Vitro Analysis of Huntingtin-Mediated Transcriptional Repression Reveals Multiple Transcription Factor Targets  Weiguo Zhai, Hyunkyung Jeong, Libin.
Paracrine Apoptotic Effect of p53 Mediated by Tumor Suppressor Par-4
Volume 24, Issue 4, Pages (July 2018)
Volume 12, Issue 6, Pages (March 2002)
Irreplaceability of Neuronal Ensembles after Memory Allocation
Volume 23, Issue 10, Pages (June 2018)
Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis  Shogo Tanabe, Toshihide Yamashita  Cell.
Presentation transcript:

Volume 22, Issue 5, Pages 1339-1349 (January 2018) IP3 Receptor-Dependent Cytoplasmic Ca2+ Signals Are Tightly Controlled by Cavβ3  Anouar Belkacemi, Xin Hui, Barbara Wardas, Matthias W. Laschke, Ulrich Wissenbach, Michael D. Menger, Peter Lipp, Andreas Beck, Veit Flockerzi  Cell Reports  Volume 22, Issue 5, Pages 1339-1349 (January 2018) DOI: 10.1016/j.celrep.2018.01.010 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Decreased Agonist-Induced Calcium Release in β3 cDNA-Expressing Cells (A and D) Confocal images of HEK (A) and Cos-7 (D) cells expressing the GFP-tagged Cavβ3 cDNA in the absence (left) and presence (right) of the α1c (Cav1.2) cDNA. (B and E) Western blot of protein lysates from HEK (B) and Cos-7 (E) cells, non-transfected or transfected with Cavβ3 cDNA (+β3). (C and F) Fura-2 ratio (F340/F380) over time before and after addition of carbachol (Cch) in HEK cells (C) or ATP in Cos-7 cells (F) in the absence of extracellular Ca2+. Control (red), cells transfected with the GFP cDNA only; +β3 (black), cells transfected with Cavβ3-IRES-GFP cDNA; (number of cells/independent experiments). Resting Ca2+ in the cytoplasm, peak amplitude, and area under the curve are presented below the traces as means ± SEM; p values were calculated using a Mann-Whitney test or unpaired two-tailed Student’s t test. See also Figures S1 and S2. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Increased Agonist-Induced Calcium Release in Cavβ3 KO Mouse Fibroblasts (A) Western blot of protein lysates from primary fibroblasts isolated from wild-type (WT) and β3 KO mice. (B) No increase in cytoplasmic [Ca2+] detectable in WT and β3 KO cells upon potassium-induced depolarization in the presence of extracellular Ca2+ (2 mM). (C and D) Fura-2 (F340/F380) ratiometric traces in the absence of extracellular Ca2+ before and after addition of bradykinin (10 μM; C) or LPA (20 μM; D) in wild-type (black) and β3 KO (red) fibroblasts (number of cells/ number of experiments). (E) Summary of resting Ca2+ in the cytoplasm, peak amplitude, and the area under the curve from experiments shown in (C) and (D). Data are presented as mean ± SEM; p values were calculated using a Mann-Whitney test. See also Figures S1 and S2. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Cavβ3 Desensitizes Cells to Low Concentrations of IP3 (A and B) Fibroblasts isolated from wild-type (WT; black) and β3 KO (red) mice (A) and Cos-7 cells (B), non-transfected (control; red) and transfected with β3 cDNA (+β3; black); cDNAs were loaded with caged IP3 and Fluo-4/AM. IP3 was released by UV flash photolysis at the indicated energies, and the cytoplasmic [Ca2+] was monitored by Fluo-4 fluorescence. Bar graph showing Ca2+ amplitude (ΔF/F0) at the indicated energies (inset, Ca2+ amplitude-energy-dose-response curves). Data are presented as mean ± SEM, and the numbers of measured cells are indicated in the bars. Both genotypes (in one dish) at a given UV light flash energy were compared; the p values were calculated using unpaired two-tailed Student’s t test. See also Figures S3 and S5. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Cavβ3 Interacts with the IP3R (A) Western blots with increasing amount of protein from wild-type and β3 KO fibroblasts were probed subsequently with antibodies for IP3R3, Na+/K+-ATPase, and β3. (B) Co-immunoprecipitation of β3 and the IP3R3. Lane 1, input (cell lysate); immunoprecipitation with the antibody for β3 (lane 2) and IP3R3 (lane 3). Western blots with antibody for IP3R3 (top) and β3 (bottom). (C) Domain structure of β3, β3ΔGK (β3-SH3[β1–β4]-HOOK-SH3[β5]) and β3ΔSH3 (β3-HOOK-SH3[β5]-GK). (D and E) Western blots after co-immunoprecipitation of IP3R3 and β3ΔGK (D) or β3ΔSH3 (E) using the antibodies as indicated. (F) Western blot confirming expression of the β3ΔSH3 cDNA in Cos-7 cells. (G) Fura-2 ratio (F340/F380) over time before and after stimulation with ATP (10 μM) in the absence of extracellular Ca2+ in Cos-7 cells expressing the β3ΔSH3-IRES-GFP cDNA (β3ΔSH3; black) and control cells expressing the GFP cDNA (red). (H) Resting cytoplasmic Ca2+, peak amplitudes, and area under the curve are shown as mean ± SEM; statistical significance was assessed using unpaired two-tailed Student’s t test for resting cytoplasmic Ca2+ and peak amplitude and Mann-Whitney test for area under the curve. See also Figure S4. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 5 The β3 Protein Reduces IP3 Binding to the IP3R (A) Western blot of microsomal proteins from wild-type (left) and β3 KO (right) mouse cerebellum using anti-β3, anti-IP3R1, and anti-β2 (lc, loading control) antibodies. (B–D) Equilibrium saturation binding of [3H]IP3 in the absence and presence of 25 μM unlabeled IP3 by cerebellum microsomes prepared from wild-type (black) and β3 KO (red) mice. (B) A representative experiment out of three is shown with Kd (C) and Bmax (D) values as indicated. Data are shown as mean ± SEM, and p values were calculated using unpaired two-tailed Student’s t test. (E and F) Coomassie-stained recombinant GST and β3-GST (E) and [3H]IP3 (10 nM) (F) binding in the absence and presence of 25 μM IP3 (non-specific binding was <40 cpm) by β3 KO cerebellum microsomes in the absence (control; 100% amounts to 0.66 ± 0.04 pmol/mg protein) and presence of recombinant β3-GST or GST as indicated. Data are shown as means ± SEM; n = 2 independent experiments in triplicate. p values were calculated using one-way ANOVA and Bonferroni’s multiple comparisons test. (G) Domain structure of IP3R indicating the suppressor domain followed by the IP3-binding domain (amino acids 1–604); six transmembrane domains are indicated in black. (H) Co-immunoprecipitation of β3 and IP3RBD (both cDNAs were co-expressed in Cos-7 cells) using anti-Cavβ3 and anti-IP3R3 antibodies for immunoprecipitation and western blot as indicated. (I) Coomassie-stained recombinant IP3BD. (J and K) Specific binding of [3H]IP3 (10 nM) (J) by recombinant IP3BD (2.50, 7.50, and 22.50 μg) in the presence of 200 nM GST protein or 200 nM GST-β3 protein. (K) Specific [3H]IP3 binding to recombinant IP3RBD (1.50 μM) in the absence (1,173.88 ± 76.48 cpm) or presence of GST and GST-β3 at increasing concentrations. Non-specific binding in the presence of 25 μM IP3 (39.40 ± 2.76 cpm) and binding to GST-β3 or GST as indicated. Data are shown as means ± SEM; p values were calculated using one-way ANOVA and Bonferroni’s multiple comparisons test. See also Figure S4. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions

Figure 6 In Vitro Fibroblast Migration and In Vivo Wound Healing (A) Representative images of migration from wild-type and β3 KO fibroblasts. (B and C) Summary of cell migration rate calculated relative to the initial scratch area after 6 hr showing increased migration of β3 KO fibroblasts (red) compared with wild-type fibroblasts (black) in the presence of 10% fetal calf serum (FCS) (B) or 1% FCS (C). Data are shown as mean ± SEM, with the experiment performed three times with three independent cell preparations (32–40 images from three independent experiments; p values were calculated using unpaired two-tailed Student’s t test). (D and E) Western blot of wild-type and β3 KO primary fibroblasts using anti-P-FAK (Sr732) (D), P-MLC (E), and anti-Akt antibody as a loading control. Bar graphs show the densitometric quantification of the intensity of the corresponding immunostain as mean ± SEM; p values were calculated using unpaired two-tailed Student’s t test. (F) Collagen secretion from wild-type (black) and β3 KO fibroblasts (red) expressed as the mean ± SEM (n = 3 independent experiments; p values were calculated using unpaired two-tailed Student’s t test). (G) Western blot of wild-type and β3 KO primary skin fibroblasts lysates (80 μg per lane) using anti-β3 antisera. (H) Images obtained from the skinfold chamber directly after wounding (day 0) and on days 3, 6, 10, and 14 post-wounding. The continuous process of wound closure, with complete epithelialization, is shown over 14 days in both genotypes. (I) At the time points indicated, the wound area was determined and plotted as a percentage of the wound area on day 0 (immediately after wounding). Data are shown as mean ± SEM (n = 8 mice; p values were calculated using two-way ANOVA and Bonferroni’s multiple comparisons test). Wound closure is significantly accelerated in β3 KO mice. See also Figure S6. Cell Reports 2018 22, 1339-1349DOI: (10.1016/j.celrep.2018.01.010) Copyright © 2018 The Authors Terms and Conditions