The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS

Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based “replace-ment” assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.Fil: Pocognoni, Cristián Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. University of Alabama at Birmingahm; Estados UnidosFil: Viktorova, Ekaterina G.. University of Maryland; Estados UnidosFil: Wright, John. University of Alabama at Birmingahm; Estados UnidosFil: Meissner, Justyna M.. University of Alabama at Birmingahm; Estados UnidosFil: Sager, Garrett. University of Alabama at Birmingahm; Estados UnidosFil: Lee, Eunjoo. University of Alabama at Birmingahm; Estados UnidosFil: Belov, George A.. University of Maryland; Estados UnidosFil: Sztul, Elizabeth. University of Alabama at Birmingahm; Estados Unido

Spectrin depletion impairs lamellipodia formation and cell adhesion.

<p><b>(A)</b> Adhesion capability of spectrin-depleted Jurkat T-cells. The data are expressed as the percentage of adherent cells compared with the number of adherent control cells. Data are presented as mean±SD for n = 3, asterisk marks statistically significant (p<0.05), Student’s t test. Abbreviations: WT-Wild type Jurkat T-cells; SC–stable cell line treated with scrambled shRNA; KD–stable cell line treated with anti-spectrin shRNA <b>(B)</b> Scanning electron microscopy of contact sites formed by Jurkat T-cells and Dynabeads coated with anti-CD3 and anti-CD28 simulating the APC activation process. Arrowheads indicate lamellipodia formed by T-cells. Magnification 20 000x, scale bar = 1 μm. <b>(C)</b> Confocal microscopy of morphological changes and actin (yellow) distribution in T-cells upon IS formation on coverslips coated with anti-CD3 and anti-CD28 antibodies. Nuclei are stained with DAPI (blue). Arrowheads indicate lamellipodia formed by T-cells. Scale bar = 5μm. <b>(D)</b> Effect of spectrin knockdown (method A of transfection) on frequency of lamellipodia formation by live-imaging of Jurkat T-cells before and after activation with Dynabeads coated with anti-CD3 and anti-CD28. Cells were co-transfected with Ruby-Life Act plasmid to visualize actin. The frequency of lamellipodia formation and actin dynamics were analyzed in the Biostation system.</p

Spectrin translocates together with actin to the IS upon T-cell activation.

<p><b>(A-B)</b> Distribution of spectrin (red, 1) and actin (green, 2) in primary T lymphocytes from PBMCs population in the absence (naive) (A) or presence (activated) (B) of Dynabeads coated with anti-CD3 and anti-CD28, along with a merged (3) and a TL(4) image. The white rectangle marks the contact site. The scale bar = 5μm. The results are representative of at least three independent experiments. <b>(C-D)</b> Transmission electron micrographs of non-activated (C) and activated with Dynabeads coated with anti-CD3 and anti-CD28 (D) Jurkat T-cells labeled with antibodies directed against spectrin, followed by gold-conjugated secondary antibodies. The right column shows enlarged images of the marked areas. Arrows point to gold particles (spectrin). Almost all of the gold in panel D accumulate at the point of cell-bead contacts. Abbreviations: APC-antigen presenting cell simulated by Dynabeads coated with anti-CD3 and anti-CD28; N-nuclei, PM-plasma membrane, M-mitochondria. Magnification 30 000x, scale bar = 1 μm (200 000x, scale bar = 200 nm), indicated on micrographs.</p

Spectrin depletion leads to impaired IS formation.

<p>Confocal images (a single z-plane) of actin (A, green), LFA-1 (B, yellow) and CD45 (C, red) distribution in wild type cells (WT) Jurkat T-cells treated with scrambled shRNA (SC) and cells treated with anti-spectrin shRNA (KD) after activation with Dynabeads coated with anti-CD3 and CD28. Fixed cells were stained with phalloidin-568, anti-LFA-1, or anti-CD45 antibodies. Asterisks indicate the location of the Dynabeads (simulating APC) in the conjugates. The scale bar = 5 μm.</p

Schematic of the immunological synapse (IS) and representative protein interactions in the synaptic space.

<p>Distribution of receptors and adhesion molecules in individual clusters in the immune synapse. The T-cell receptor (TCR) / CD3 complex interacts with MHC-peptide. The adhesion molecules on the surface of both cells (LFA-1—ICAM- 1 are responsible for the formation and stabilization of the IS, as well as for initiating signal transduction pathways activated by TCR. The distal ring of IS is rich in proteins, such as CD45 and F-actin controls lamellipodia and filopodia formation.</p

Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based “replace-ment” assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.Fil: Pocognoni, Cristián Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. University of Alabama at Birmingahm; Estados UnidosFil: Viktorova, Ekaterina G.. University of Maryland; Estados UnidosFil: Wright, John. University of Alabama at Birmingahm; Estados UnidosFil: Meissner, Justyna M.. University of Alabama at Birmingahm; Estados UnidosFil: Sager, Garrett. University of Alabama at Birmingahm; Estados UnidosFil: Lee, Eunjoo. University of Alabama at Birmingahm; Estados UnidosFil: Belov, George A.. University of Maryland; Estados UnidosFil: Sztul, Elizabeth. University of Alabama at Birmingahm; Estados Unido