Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

<div><p>Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase.</p></div

SNAP-23 is essential for B cell survival.

<p>Spleens were harvested from CD19-Cre⁺ SNAP-23^fl/+ control mice or CD19-Cre⁺ SNAP-23^fl/- mice (line E3) and single cell suspensions were stained with fluorochrome-conjugated CD19- and CD3-mAb and analyzed by flow cytometry. (A) A representative flow cytometry profile reveals a dramatic reduction in the percentage of CD19⁺ B cells in the spleen. (B) Quantitative analysis of two different matched pairs of CD19-Cre⁺ SNAP-23^fl/+ or SNAP-23^fl/- littermate mice indicating total spleen cell numbers as well as the numbers of T cells (CD3⁺ cells) and B cells (CD19⁺ cells) present in each spleen (calculated based on the percentage of each cell type present as determined by flow cytometry). (C) Summary of three independent experiments showing the recovery of the indicated cell types from the spleens of CD19-Cre⁺ SNAP-23^fl/- mice expressed as a percentage of those found in CD19-Cre⁺ SNAP-23^fl/+ control mouse spleens. The data shown are mean +/- SD of three independent experiments (*p<0.05). (D) B cells were purified from the spleens from the indicated mice and equal numbers of cell equivalents were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control.</p

Generation of mice containing a floxed SNAP-23 allele.

<p>(A) A 179 kb BAC clone containing the entire SNAP-23 gene was mutated by introducing loxP sites surrounding exon II alone (#1 floxed allele) or surrounding exons III-V (#2 floxed allele). Expression of Cre will result in the excision of exon II alone in the #1 floxed allele construct or exons III-V in #2 floxed allele construct. Each deleted allele introduces a frame shift that prevents the synthesis of in-frame SNAP-23 exon-encoded amino acids. (B) Typical PCR genotyping analysis of mice used in this study. The upper panel shows results obtained using a PCR reaction that can distinguish the deleted SNAP-23 allele present in SNAP-23^+/- mice (492 bp) from either the wild-type SNAP-23 allele or the floxed SNAP-23 BAC transgene (266 bp). The lower panel shows results obtained using a PCR reaction that can distinguish the floxed SNAP-23 BAC transgene (195 bp) from the wild-type SNAP-23 allele present in SNAP-23^+/- mice (161 bp). (C) Spleen cells were isolated from wild-type mice (SNAP-23^+/+) and two representative floxed SNAP-23 BAC transgene mice on a SNAP-23-/- background (SNAP-23^fl/-) and the cells were lysed and analyzed by SDS-PAGE and immunobloting with SNAP-23 and actin antibodies. Quantitative densitometry revealed that the floxed SNAP-23 BAC expression of SNAP-23 was approximately 50% that found in wild-type mice.</p

Deletion of SNAP-23 leads to acute death of MEFs.

<p>MEF lines were generated from SNAP-23^fl/+ mice (clones 75.1 and 88.2) or SNAP-23^fl/- mice (clones 75.2 and 88.3). (A) Infection of a typical SNAP-23^fl/+ MEF culture infected with empty retrovirus (left panel) or GFP-Cre retrovirus (right panel) shows that two days after infection the vast majority of MEFs were viable (Annexin V^-) and expressed GFP-Cre. (B) The indicated MEF lines were infected with GFP-Cre-expressing retrovirus and the number of live cells present in each culture (based on staining with PI) was determined at different times. The absolute cell recovery in each condition was expressed relative to the amount of cells present two days after infection (control experiments showed that there was no Cre-dependent cell death in any line after only two days of infection). The data shown are representative of two independent experiments analyzed at day 2, 4, 6, 8 and one experiment analyzed at day 1, 3, 5, 7. (C) Adherent SNAP-23^fl/- MEFs were isolated 4 days after retroviral transduction with Cre (Cre+) or after mock-transduction (Cre-). Equal numbers of cells from each culture were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control. The amount of SNAP-23 present in each cell lysate was normalized to the amount of actin present and the data shown are mean +/- SD of three independent experiments (*p<0.05). (D) MEF lines generated from SNAP-23^fl/+ mice or SNAP-23^fl/- mice were co-infected with retrovirus containing GFP-Cre and retrovirus containing nothing (mock), mouse SNAP-23 (mSNAP-23), or human SNAP-23 (hSNAP-23). After 4 days the number of live cells present in each culture (based on staining with PI) was determined and was expressed relative to the number of cells recovered in the mSNAP-23 co-infection condition. The data shown are mean +/- SD of four independent experiments (*p<0.05). (E) The indicated MEF lines were infected with GFP-Cre-expressing retrovirus and after four days the cells were stained with PI and Annexin V and analyzed for GFP-Cre expression. The percentage of viable (PI^-) GFP-Cre⁺ Annexin V⁺ cells in each culture was determined by flow cytometry. The data shown are representative of three independent experiments.</p

Deletion of SNAP-23 in Lck-expressing T cells prevents T cell development.

<p>Thymi were harvested from Lck-Cre⁺ SNAP-23^fl/+ control mice or Lck-Cre⁺ SNAP-23^fl/- mice and single cell suspensions were stained with fluorochrome-conjugated CD4- and CD8-mAb and analyzed by flow cytometry. (A) A representative flow cytometry profile reveals a dramatic reduction in the percentage of CD4⁺, CD4⁺8⁺, and CD8⁺ T cells in the thymus of an Lck-Cre⁺-expressing SNAP-23^fl/- mouse (line D2). (B) Quantitative analysis of matched pairs of littermate mice from SNAP-23^fl founder lines E3 or D2 indicate recovery of CD4^-CD8^- (DN), CD4⁺CD8⁺ (DP), CD4⁺, and CD8⁺ T cells present in thymi of Lck-Cre⁺ SNAP-23^fl/- mice expressed as a percentage of those found in thymi of Lck-Cre⁺ SNAP-23^fl/+ control mice (calculated based on the percentage of each cell type present as determined by flow cytometry). The data shown are mean +/- SD of three independent experiments (*p<0.05). (C) The recovery of the indicated cell types from the spleens of Lck-Cre⁺ SNAP-23^fl/- mice was expressed as a percentage of those found in Lck-Cre⁺ SNAP-23^fl/+ control mouse spleens (line D5). The data shown are mean +/- SD of eight independent experiments (*p<0.05). (D) T cells were purified from the spleens from the indicated mice and equal numbers of cell equivalents were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control.</p

<i>Snap23</i>^Δ/Δ blastocysts die prior to uterine implantation.

<p>(<b>A</b>) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated <i>Snap23</i>^Δ/wt females mated with <i>Snap23</i>^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018444#pone-0018444-t002" target="_blank">Table 2</a>). (<b>B</b>) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the <i>Snap23</i> deleted allele (<i>Snap23</i>^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the <i>Snap23</i>^wt allele (266 bp) and for the <i>Snap23</i>^Δ allele (492 bp) are indicated.</p

Genotype analysis from timed-pregnant matings of <i>Snap23</i>^Δ/wt heterozygote mice.

<p>Embryo age, number of dissected embryos, and <i>Snap23</i> genotype results are summarized as indicated.</p

Expression of SNAP-23 protein is reduced by half in <i>Snap23</i>^fl/wt and <i>Snap23</i>^Δ/wt mice.

<p>(<b>A</b>) <i>Snap23</i>^fl/wt heterozygous mice were mated and twelve two-week old pups from this mating were genotyped and analyzed for SNAP-23 protein expression. Genotyping of tail DNA was performed using PCR Primer set 2 from tail DNA. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 400 bp) is present in <i>Snap23</i>^fl/wt pups. No homozygous <i>Snap23</i>^fl/fl pups were obtained when more than 50 pups were analyzed from <i>Snap23</i>^fl/wt heterozygous matings. For immunoblot analysis, whole brain was solubilized in modified RIPA lysis buffer and protein levels were analyzed by immunoblotting (WB) as indicated antibodies. (<b>B</b>) <i>Snap23</i>^Δ/wt heterozygous mice were mated and pups from this mating were genotyped and their brains were analyzed for expression of SNAP-23 and other SNARE proteins. Genotyping of tail DNA was performed using PCR Primer set 2. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 492 bp) is present in <i>Snap23</i>^Δ/wt pups. No homozygous <i>Snap23</i>^Δ/Δ pups were ever obtained from <i>Snap23</i>^Δ/wt heterozygous matings. Whole brains were solubilized and analyzed by immunoblotting (WB) using the indicated antibodies.</p

Oligonucleotide primers used in this study and estimated size of PCR products.

<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Snap23</i>, and to generate probes for Southern blot analysis. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p

MARCH1Δ and Class II K225R BMMØ Fail to Down-Regulate Class II in Response to FTMØSN.

<p>IFN-γ activated wild type (WT), MARCH1Δ (M1Δ) or class II K225R (K>R) BMMØ were treated with mock SN or FTMØSN. After 20–24 hours, total MØ class II (II) and GAPDH (G) levels were determined by western blot analysis of whole cell lysates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037330#pone.0037330-Wilson1" target="_blank">[7]</a>. Shown are representative results from 1 of 3 independent experiments.</p