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

First published online 30 October 2007
doi: 10.1242/jcs.011213

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Repass, S. L. Articles by O'Halloran, T. J. Search for Related Content PubMed PubMed Citation Articles by Repass, S. L. Articles by O'Halloran, T. J.

Dictyostelium Hip1r contributes to spore shape and requires epsin for phosphorylation and localization

Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin TX 78712, USA

View larger version (11K): [in this window] [in a new window]   Fig. 1. Domain structures of Hip1r orthologs in different species. Like other Sla2/Hip1 family members, Dictyostelium Hip1r contains an ANTH domain, a central coiled-coil region and THATCH domain as well as proline- and glutamine-rich regions. Over its entire length, Hip1r shares 24% identity with Sla2p, and 20% and 19% identity with mouse and human HIP1R, respectively. Overlap of shaded regions indicates areas of overlap between domains. Dicty, Dictyostelium discoideum; Yeast, Saccharomyces cerevisiae; Mouse, Mus musculus; Pro, proline; Gln, glutamine.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Hip1r is a phosphorylated protein that associates with membrane and Triton-X100-insoluble fractions. (A) Subcellular cell fractionation of wild-type cells. Most of the cellular pool of Hip1r associates with large membrane structures (LSPs), whereas the rest is soluble (HSS). (B) Triton-X fractionation of Hip1r. Cells were resuspended into buffer containing 0.5% Triton-X100 and fractionated, resulting in a portion of Hip1r sedimenting with the insoluble fraction (P). Extraction of the detergent-insoluble pellet with high-salt buffer shifts Hip1r to the soluble fraction (S). (C) Hip1r is phosphorylated in vivo. Cells were lysed and incubated with calf intestinal phosphatase (CIP) for 25 minutes, resulting in the disappearance of the upper band of the Hip1r doublet. The control lane includes CIP plus okadaic acid, a phosphatase inhibitor. WCL, whole-cell lysate; LSP, low-speed pellet; LSS, low-speed supernatant; HSP, high-speed pellet; HSS, high-speed supernatant; S, supernatant; P, pellet; CIP, calf intestinal phosphatase-treated sample; control, CIP plus okadaic acid.

View larger version (85K): [in this window] [in a new window]   Fig. 3. Hip1r-null mutants develop normally into fruiting bodies. Wild-type and Hip1r-null cells were harvested and inoculated onto non-nutrient agar plates to induce the Dictyostelium developmental program. (A,B) Both wild-type (WT) and Hip1r-null cells (Hip1r-) produce fruiting bodies consisting of a round sorus, supported by a thin stalk. Bar, 0.25 mm. (C,D) The stalks from wild-type (WT) and Hip1r-null cells are similar in size and cellular organization. Bar, 10 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 4. Dictyostelium Hip1r-null cells produce round, calcofluor-positive spores that have SP70 within the spore coats. (A-D) Wild-type and Hip1r-null spores were harvested, stained with the cellulose-staining dye calcofluor and viewed by phase-contrast or fluorescence microscopy. Wild-type (WT) spores are elliptical in morphology, whereas Hip1r-null spores (Hip1r-) are round. Bar, 5 µm. (E-T) Hip1r-null cells secrete outer-layer spore coat proteins. Wild-type (WT) and Hip1r-null spores were harvested, fixed and stained with an antibody against an outer spore coat protein, SP70, and viewed by DIC (E-H and M-P) or fluorescence (I-L and Q-T) microscopy. Bar, 5 µm.

View larger version (181K): [in this window] [in a new window]   Fig. 5. Hip1r-null spore coats lack organized cellulose. Wild-type and Hip1r-null spores were collected, fixed and stained (see Materials and Methods). (A,B) Wild-type (WT) and Hip1r-null (Hip1r-) spores were collected and washed without detergent. Wild-type spores are elliptical, whereas null spores are round (C,D), and washing wild-type spores and Hip1r-null spores with 0.1% NP40 before fixation results in the collapse of the mutant spores. Bars, 5 µm. (E,F) TEM of spores shows a middle layer of protein arranged in fibrils or crosslinked in wild-type (arrows) spores that is absent for the spore coat of mutant spores. Bar, 500 nm.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Hip1r epsin double-mutant cells display developmental phenotypes similar to those of Hip1r single-mutant cells. Wild-type (WT), epsin-null (epsin-), Hip1r-null (Hip1r-) and Hip1r epsin (Hip1r-/epsin-) double-mutant cells were harvested and inoculated onto non-nutrient agar plates to induce spore formation. Spores were examined by DIC microscopy. Wild-type spores (A) are ovoid, whereas epsin-null spores (B) are less oblong, with some round spores. Hip1r-null spores (C) and Hip1r epsin double-null spores (D) are completely round. Bar, 5 µm. (E) When treated with detergent or heat, Hip1r-null and Hip1r epsin double-mutant spores show decreased viability. Wild-type, epsin-null, Hip1r-null and Hip1r epsin double-mutant spores were harvested and left untreated, subjected to washes with the detergent NP40 or heat treated for 10 minutes. Spores were then plated on a bacterial lawn, where they germinated and grew. Cleared plaques were counted as a measure of spore germination. Error bars represent the s.e. (n=1000 spores, seven experiments).

View larger version (101K): [in this window] [in a new window]   Fig. 7. Hip1r localizes to the plasma membrane and cytoplasm and displays an interaction with epsin. Growing cells were fixed and stained with antibody against Hip1r. (A) Cells were viewed by DIC microscopy. (B) Cells stained with antibody against Hip1r show bright Hip1r puncta at the cell cortex when viewed by fluorescence microscopy. (C-E) Focusing on the surface of cells expressing a clathrin-GFP construct (clathrin:GFP) shows partial colocalization of clathrin (green) with Hip1r (anti-Hip1r, red). Examples are indicated by arrowheads (E). (F-H) Cells expressing an epsin-GFP fusion protein (epsin:GFP, green) and stained with antibody against Hip1r (anti-Hip1r, red) exhibit extensive colocalization at the plasma membrane (arrowheads). Occasionally, puncta contain epsin but not Hip1r (arrow). Bar, 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 8. The N-terminal ENTH domain of epsin is necessary and sufficient for phosphorylation of Hip1r and membrane localization. (A,B) In wild-type (A, WT) and Hip1r-null cells (B, Hip1r-), epsin-GFP (epsin:GFP) localizes within puncta on the plasma membrane. (C,D) In contrast to wild-type cells (C), Hip1r puncta (anti-Hip1r) are not restricted to the cell periphery in epsin-null cells (D, epsin-). (E) Expression of the ENTH domain in epsin-null cells [epsin-(ENTH:GFP)] restores the peripheral localization of Hip1r. Bar, 10 µm. (F) Epsin influences the phosphorylation state of Hip1r. Immunoblots of cell lysates of WT cells, epsin-null cells expressing epsin-GFP (epsin-, epsin:GFP) and the ENTH domain of epsin (epsin-, ENTH:GFP) probed with antibody against Hip1r exhibit double bands, indicative of phosphorylation of Hip1r. In epsin-null cells (epsin-) and cells expressing only the epsin C-terminus (epsin-, C-term), Hip1r is not phosphorylated.