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Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment

Rodrigo Bustos^1,*, E. Robert Kolen^1,*, Lelita Braiterman¹, Anthony J. Baines², Fred S. Gorelick³ and Ann L. Hubbard^3,

¹ Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2105, USA
² Biological Laboratory, University of Kent, Kent, CT2 7NJ, UK
³ Division of Gastroenterology, Department of Medicine, Yale University School of Medicine, West Haven, CT 06516, USA
^* These authors contributed equally to this work

View larger version (75K): [in a new window]   Fig. 1. Synapsin I is present in non-neuronal cells. (A,B) Immunoblot analysis of total homogenates of rat brain (1 µg), rat liver (200 µg), rat hepatocytes (Hep, 200 µg) and NRK cells (200 µg) using antibody SNH1 (A) and SNT1 (B). Both antibodies detected an 85 kDa band (*) in non-neuronal samples that co-migrated with rat brain synapsin I. Molecular mass standards (in kDa) are indicated at the left of each panel. (C) Antibodies to synapsin I immunoprecipitate an 85 kDa protein from liver. Proteins from brain (0.5 mg total protein) or liver (10 mg total protein) cytosols were immunoprecipitated with SNH1 (top panel) or SNT1 (bottom panel) and analyzed by immunoblot with Ab#213. Both antibodies immunoprecipitated an 85 kDa protein from either brain (Brain +ab) or liver cytosol (Liver +ab) that was recognized by Ab#213; the protein was not immunoprecipitated from liver cytosol incubated with Protein A-Sepharose beads alone (Liver -ab). (D) The 85 kDa protein is absent from livers of synapsin I- and II-deficient mice. Total homogenates of brain or liver from wild-type mice (wt) or synapsin I and II-deficient mice (syn-/-) were analyzed by immunoblot using Ab#212 (SynI) or antibodies to tubulin (Tub) as a loading control. An 85 kDa protein was detected in both brain and liver of wild-type mice that was absent from brain and liver of synapsin I and II-deficient mice.

View larger version (44K): [in a new window]   Fig. 2. Synapsin I from NRK cells and rat brain show similar phosphopeptide patterns. (A) Synapsin I immunoprecipitated from NRK cells was phosphorylated in vitro by PKA. By autoradiography, a phosphorylated species was detected that co-migrated with rat brain synapsin I and was labeled by SNT1 in immunoblot (*, lanes 1,4). The signal was absent if the anti-synapsin I antibody or the NRK extract were absent during the immunoprecipitation (lanes 2,3). (B) The PKA-phosphorylated proteins were digested with V8 endoprotease or chymotrypsin, the fragments were resolved on a 15% gel, transferred to nitrocellulose and exposed to autoradiographic film. The phosphopeptide maps for both synapsin I purified from rat brain and synapsin I immunoprecipitated from NRK cells are identical. Position of molecular mass standards (in kDa) is indicated.

View larger version (85K): [in a new window]   Fig. 3. Detection of synapsin I mRNA from freshly isolated hepatocytes by northern blot. Membrane was probed with a 3' end probe from rat brain synapsin I. In brain total RNA (2 µg, lanes 1,2), the synapsin I message was detected as two species (*), at 3.3 and 4.3 kb. The synapsin I mRNA was detected in hepatocytes total RNA (22 µg, lane 3) and poly(A) RNA (3.3 µg, lane 4) (** and ***) at 2.5-3.1 kb. Presence of synapsin I message has been confirmed in three independent preparations of hepatocyte poly(A) RNA. Lane 1, 10 day exposure; lanes 2-4, 5 day exposure. Position of size standards is indicated in kb. The square bracket indicates 28S rRNA.

View larger version (74K): [in a new window]   Fig. 4. Synapsin I localizes to the Golgi area of NRK cells. Cells were double immunostained for synapsin I (SynI) (A,a, B,b) and mannosidase II (mann II) (A’,a’) or TGN38 (B’,b’). Boxed regions in A, A’,B,B’ are shown enlarged in a,a’,b,b’, respectively. Asterisks mark cell nuclei. Bar, 10 µm. (C) Confocal sections acquired at different levels of the Golgi of a NRK cell double-stained for synapsin I (green) and TGN38 (red). Consecutive sections are 0.5 µm apart. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 5. Synapsin I remains associated with the Golgi in cells exposed to cytoskeletal-disrupting agents. (A) Nocodazole treatment causes synapsin I to disperse in punctate structures throughout the cytoplasm, in a similar manner to TGN38. NRK cells were treated with 33 µM nocodazole for 1 hour at 37°C, then fixed and stained for tubulin or double stained for synapsin I and TGN38. Tubulin staining shows complete microtubule depolymerization in nocodazole-treated cells. (B) Cytochalasin D treatment has no effect on synapsin I distribution in NRK cells. NRK cells were treated for 2 hours with 5 µM cytochalasin D. FITC-phalloidin staining reveals F-actin disruption in cytochalasin D-treated cells. Staining for synapsin I reveals punctate staining at cell center, similar to untreated cells. Asterisks mark cell nuclei. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 6. Colocalization studies between synapsin I and different markers in NRK cells. NRK cells were double stained with SNT1 (A,a,B,b,C,c,D,d,E,e) and antibodies against adaptin (A’,a’); the transferrin receptor (Tf R) (B’,b’); ßCOP (C’,c’); syntaxin 6 (D’,d’); and SCAMP4 (E’,e’). Boxed regions in A,A’,B,B’,C,C’,D,D’,E,E’ are shown enlarged in a,a’,b,b’,c,c’,d,d’,e,e’, respectively. Synapsin I staining localizes to the juxtanuclear region of the cells as well as staining for the other markers. However, in all cases, the synapsin I compartment does not appear to be stained by the second antibody. Asterisks mark cell nuclei. Bar, 10 µm.

View larger version (99K): [in a new window]   Fig. 7. Synapsin I and myosin II colocalize at the Golgi region in NRK and WIF-B cells. Methanol-fixed NRK (A,A’,a,a’) and WIF-B (B,B’,b,b’) cells were double-stained for synapsin I (A,a,B,B) and myosin II (A’,a’,B’,b’). Both antigens localize to the same juxtanuclear structures in these cells. Boxed regions in A,A’,B,B’ are shown enlarged in a,a’,b,b’, respectively. Non-Golgi staining of myosin II antibody is due to labeling of stress fibers. Asterisks mark cell nuclei. Stars mark an apical cyst enclosed by two polarized WIF-B cells. Bar, 10 µm.