QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pullikuth, A. K. Articles by Gill, S. S. Search for Related Content PubMed PubMed Citation Articles by Pullikuth, A. K. Articles by Gill, S. S.

In vivo membrane trafficking role for an insect N-ethylmaleimide-sensitive factor which is developmentally regulated in endocrine cells

Ashok K. Pullikuth¹ and Sarjeet S. Gill^1,2,*

¹ Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA
² Graduate Programs in Environmental Toxicology, Genetics, Biochemistry and Molecular Biology, University of California, Riverside, CA 92521, USA

View larger version (34K): [in a new window]   Fig. 3. Restoration of intra-Golgi and post-Golgi trafficking by MsNSF expression in vivo. (A) Cells were grown in induction medium for 4 h, shifted to 32°C for 30 min and labeled for 30 min. Cell and medium portions were separately precipitated with TCA and analyzed by immunoprecipitation with anti- factor antibody. After 30 min of chase time at 32°C, cells were shifted to 24°C to follow the blocked forms to maturation. Cells were derived from EGY1181-5 (sec18-1) (Table 1), ASHY1181-1 (vector only) (lanes 1-4), and ASHY 1181-2 (PGAL-MsNSF) (lanes 5-8). During 0 min chase a substantial amount of -factor already resides in the Golgi (lane 5) in MsNSF cells, and this is rapidly matured and secreted (lane 6, 30min chase). In sec18-1 cells the ER form predominates all through the chase time (compare lanes 1 and 2), and is only processed more slowly when returned to the permissive temperature (30 and 60 min chase at 24°C, lanes 3 and 4). The mature peptide was not immunoprecipitated with this antibody. Thus maturation of -factor is inferred from the disappearance of the Golgi (G)-modified form during the chase. ER, G and pG denote the expected positions of ER, Golgi and post-Golgi forms of -factor. The positions of molecular mass markers (kDa) are shown on the left. (B) Analysis of proteins secreted into the medium at restrictive temperatures. Expression of MsNSF in sec18-1 mutants restores transport of proteins secreted into the medium at the restrictive temperature of 37°C. The band at 150 kDa corresponds to an extensively O-glycosylated heat shock protein, HSP150. Wild-type (RSY 248, lane 1) or cells derived from sec18-1 (as mentioned in Fig. 1) were incubated at 37°C for 15 min, labeled and chased for 30 min each at 37°C. Arrows indicate seven predominant proteins that are secretion-blocked in sec18-1 cells. Medium from 0.5 A600 cell equivalents was analyzed by autoradiography. Cell pellets were immunoprecipitated with MsNSF antibody to confirm the expression of MsNSF (bottom). (C) Immunoblot of secreted HSP150 at 37°C. Cells were grown overnight in rich medium (supplemented with 3% galactose) to a density of 1 A600 unit ml-1. Cells (25 A600 units) were concentrated and washed twice in water and once in medium and resuspended in prewarmed medium (37°C) to a density of 10 A600 units ml-1. Cells (1 ml) were incubated at 37°C for 30 min and equal portions (0 min chase, odd-numbered lanes) were transferred to NaN3-NaF (20 mmol l-1) on ice. The remainder of the culture was washed in prewarmed medium to remove pre-existing proteins and resuspended in prewarmed medium containing cycloheximide (20 µg ml-1); proteins were chased for 30 min (even-numbered lanes). Samples equivalent to 3 A600 units of cells were analyzed by immunoblotting with HSP150 antibody. WT, wild type.

View larger version (39K): [in a new window]   Fig. 1. In vivo expression of MsNSF rescues the temperature-sensitive growth defect in sec18-1 yeast mutant. (A) Wild-type (RSY 248) or strains derived from sec18-1 (SEY 5186) (Table 1) containing the `empty' vector, pYES2 (ASHY1896-1), wild-type SEC18 gene (ASHY1896-5) or SEC18 (ASHY1896-7) and MsNSF (ASHY1896-2) cloned downstream from the GAL1 promoter (PGAL) were grown at 24 °C. Serial dilutions (indicated above panels) were spotted on to rich agar medium containing 3 % galactose and incubated at 24 °C (left), 30 °C (middle) or at 37 °C (right) for 3 days. (B) Cells were incubated at 32 °C in rich agar medium containing 3 % galactose (left), 2 % raffinose (middle) or 5 % glucose (right) as carbon source. (C) Wild-type (X2180-1B) or cells derived from sec18-1 containing various constructs were incubated at 37 °C in a humid chamber for 8 days and returned to 22 °C for 4 days.

View larger version (52K): [in a new window]   Fig. 2. Progression and maturation of CPY at non-permissive temperature is promoted by MsNSF. (A) Expression of MsNSF in sec18-1 removes the transport block for CPY. Cells were grown to a density of 1 A600 unit ml-1 in minimal medium lacking methionine and uracil, supplemented with 3 % galactose and 200 µmol l-1 (NH4)2SO4 for 2 h (A) or 6 h (B,C) to induce expression. Cells were resuspended in fresh medium (lacking sulfate), incubated at 33 °C for 30 min and labeled with trans-35S for 30 min at 33 °C. Cells (10 A600 units) were removed at the indicated time intervals and analyzed by CPY immunoprecipitation (IP). Wild-type (B) and MsNSF (C) cells were induced for 6 h before temperature-shift and pulse-chase, and CPY IP was done as in A. Images from three independent experiments were digitized from various exposures and analyzed by NIH image. The CPY maturation index (MI) was calculated from quantified values as [mCPY/(p1+p2+mCPY)]x100. The coefficient of variation was <6 %. Also shown in C is MsNSF (83 kDa) immunoprecipitated with -MsNSF antibody after CPY-IP. The strains used are the same as in Fig. 1 (see Table 1 for genotypes). The relevant portions of autoradiographs are shown. p1 (67 kDa), p2 (69 kDa) and mCPY (61 kDa) refer to ER, Golgi and vacuolar forms of CPY, respectively.

View larger version (72K): [in a new window]   Fig. 4. Expression and localization of M. sexta NSF (MsNSF). (A) Expression of MsNSF is enriched in neuronal tissues of larvae, pupa and developing adults. Midgut (MG), hindgut (HG) and Malpighian tubules (MT) contain reduced reactive species. Detergent-solubilized protein extracts (50 µg lane-1) were separated by SDS-PAGE and analyzed by immunoblotting with affinity-purified -MsNSF antibodies. The positions of molecular mass markers (kDa) are shown. Neonate larvae (B-D) or fifth-instar midguts (E,F) were stained with -FMRFamide (B,C) or -MsNSF antibodies (D-F) and detected by Cy3-conjugated secondary antibodies. Enteric neurons and their axons and a large set of enteric endocrine cells could be marked by FMRFamide reactivity (B,C). Enriched MsNSF immunoreactivity (eNSF-IR) is not present in any enteric neurons or their axons. Six rows of enteric endocrine cells express eNSF-IR only in the median midgut (D), while staining in regions anterior and posterior was unremarkable (not shown). Pediculate cells of midgut stain for MsNSF either singly (F) or in twos (F, inset) per invagination. MsNSF expression predominates in the basolateral membrane of globet cells (E) and plasma membrane of Malpighian tubules (G,I). MsNSF expression is also detected in the glomeruli (g; H) in the antennal lobe of adult and synaptic boutons of larval protocerebrum (J). (K-M) MsNSF exhibits enrichment in photoreceptors, structures of the optic lobe and the antennal lobe of M. sexta. MsNSF is expressed in photoreceptors, mostly localizing to the cytoplasm (M) and granular structures (K,L) of the optic lobe. Lamina (La), medulla (Me), lobula (Lo) and lobula plate (Lp) intensely stain for MsNSF, indicating that MsNSF might function in the visual system of M. sexta. Whole-mount (B-D,J) and paraffin section histochemistry were performed as described in Materials and methods.

View larger version (37K): [in a new window]   Fig. 5. Expression of MsNSF is developmentally regulated in a subset of neuroendocrine cells and is selectively enriched in specific subsites of hormonal release in M. sexta brain. Enriched MsNSF immunoreactivity (eNSF-IR) is present on or after day 3 of pupation and continues throughout adult development (B-E). FMRFamide staining detects most cells of the neurosecretory complex and various varicosities in the brain (F). (G) Paraffin section showing exaggerated Type I staining and eNSF-IR (arrow). The image was overexposed to show both eNSF-IR and Type I staining. eNSF-IR is present only in the corpora cardiaca (CC) (H,I) and excluded from the corpora allata (CA). (I) A higher magnification of eNSF-IR in varicosities of CC in neurohemal complex. Note that no other neurosecretory cells are stained in paraffin sections (G), ruling out problems of antibody penetration in our whole mounts. A representative staining with pre-immune IgG is shown in A, for comparison of specific signals with background staining.

View larger version (61K): [in a new window]   Fig. 6. Novel, developmentally regulated expression of MsNSF. Pharate adult brains were analyzed by staining with antibodies against MsNSF (A,C,F), diuretic hormone (DH) (B,C), eclosion hormone (EH) (D,E) or bombyxin (A). Bombyxin is synthesized in four pairs of cells in the anterior protocerebrum (A, green) that do not colocalize with enriched MsNSF immunoreactivity (A, red). Diuretic hormone (DH) in later stages is localized to 80-100 neurosecretory cells (B), which lie medial to the eNSF-IR cells, as revealed by costaining with DH and MsNSF antibodies (C, arrows). EH in larvae is synthesized by paired ventro-medial cells (D). In later stages of development, EH-containing ventromedial cells in the brain migrate anteriorly, to the proctocerebrum, and are positioned ventrally to the eNSF-IR cells (E). Axons of eNSF-IR run parallel to EH axons to end in the CC—CA complex (F). The posterior aspect of brains stained for EH (E) or MsNSF is shown (F). Note the axons of eNSF-IR from Type IIa4 cells run ventrally from the dorsally situated cell bodies (outside the plane of focus, arrow) in F; asterisks denote Type I staining.