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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01608

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Nak1 interacts with Hob1 and Wsp1 to regulate cell growth and polarity in Schizosaccharomyces pombe

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 4N1, Canada

View larger version (37K): [in a new window]   Fig. 1. Hob1 interacts with Nak1. Epitope-tagged proteins were expressed in wild-type (RL143) cells using nmt1 promoter expression plasmids, and were assayed for expression by western-blot analysis using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (top). Extracts were immunoprecipitated with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (bottom). (A) Control vectors (lane 1), Myc-Nak1 alone (lane 2), HA-Hob1 and Myc-Nak1 (lane 3), HA-Hob1 alone (lane 4), and HA-Hob1 and Myc-Nak1-562 (lacking the CTR; lane 5). (B) Control vectors (lane 1), HA-Nak1 (lane 2), HA-Nak1 and Myc-Hob1 (lane 3), HA-Nak1 and Myc-Hob11-281 (lane 4), Myc-Hob1 (lane 5), and Myc-Hob11-281 alone (lane 6).

View larger version (29K): [in a new window]   Fig. 2. Nak1 and Hob1 interact with Wsp1. Extracts (top) and anti-HA immunoprecipitates (bottom) from wild-type (RL143) cells expressing HA- or Myc-tagged proteins from nmt1 promoter expression plasmids were assayed by western-blot analysis using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies. (A) Control vectors (lane 1), Wsp1-HA alone (lane 2), Wsp1-HA and Myc-Hob1 (lane 3), and Myc-Hob1 alone (lane 4). (B) Control vectors (lane 1), Myc-Wsp1 (lane 2), HA-Hob1 and MycWsp1 (lane 3), HA-Hob11-281 and Myc-Wsp1 (lane 4), and HA-Hob1275-466 and Myc-Wsp1 (lane5). (C) Control vectors (lane 1), Myc-Wsp1 alone (lane 2) and Myc-Wsp1 and HA-Nak1 (lane 3).

View larger version (88K): [in a new window]   Fig. 3. Hob1 localization to sites of cell growth and division requires Nak1. (A) IRG2 (hob1-GFP) cells were grown to mid-log phase in selective medium, stained with calcofluor (left) or TRITC-phalloidin (right) and examined by fluorescence microscopy; panels on the right are enlarged. DY201 (cdc10 hob1-GFP) and DY202 (cdc25 hob1-GFP) cells were grown at 25°C to log phase and then shifted to 36°C for 4 hours to block cells before and after NETO, respectively (bottom, cdc10 and cdc25). (B) IRG7 (hob1-GFP nmt1-nak1) cells were grown in the absence (+Nak1) or presence (–Nak1) of 100 µg ml–1 thiamine for 18 hours to repress endogenous nak1 expression. IRG7 cells expressing Nak1T171A and Nak11-562 from adh1 promoter expression plasmids were also grown to repress endogenous nak1 expression (top right). WIR1 (hob1-GFP nmt1-wsp1) cells were grown in the absence (+Wsp1) or presence (–Wsp1) of 100 µg ml–1 thiamine for 18 hours to repress endogenous wsp1 expression.

View larger version (55K): [in a new window]   Fig. 4. Wsp1 localization to sites of cell growth requires Nak1. (A) WSP3-12B (wsp1) cells expressing Wsp1-GFP from an adh1 promoter expression plasmid were grown to mid-log phase in minimal medium and stained with TRITC-phalloidin (top) or calcofluor (bottom), and examined by fluorescence microscopy. (B) TYH1 (nmt1-nak1) cells expressing Wsp1-GFP were grown in the absence (+Nak1) or presence (–Nak1) of 100 µg ml–1 thiamine for 18 hours to repress endogenous nak1 expression. TYH1 (nmt1-nak1) cells coexpressing Wsp1-GFP and Nak1T171A (+Nak1T171A) were also examined following endogenous nak1 repression.

View larger version (96K): [in a new window]   Fig. 5. Localization of Hob1, but not Wsp1, depends on F-actin integrity. (A) IRG2 (hob1-GFP) cells (Hob1-GFP and actin panels) or SPU (wild type) cells expressing GFP/-tubulin (microtubules) were grown to mid-log phase and incubated for 15 minutes in the presence of 1% dimethyl sulfoxide (DMSO) (top), 20 µM LatA and 1% DMSO (LatA, middle) or 100 µM TBZ and 1% DMSO (TBZ, bottom). Some cells were then fixed and stained with TRITC-phalloidin to observe actin localization. (B) Cells expressing Wsp1-GFP were similarly grown to mid-log phase, treated with DMSO, LatA or TBZ, and examined for Wsp1-GFP localization.

View larger version (53K): [in a new window]   Fig. 6. Hob1 overexpression in wild-type and nak1-repressed cells. (A) Wild-type RL143 cells containing a control plasmid or expressing HA-Hob1, HA-Hob11-281 or HA-Hob1275-466 from adh1 promoter expression plasmids were grown on selective medium at 30°C for 3 days. (B) TYH1 (nmt1-nak1) cells containing a control plasmid or expressing HA-Nak1, HA-Hob1, HA-Hob11-281 or HA-Hob1275-466 were grown on minimal medium plus 100 µg ml–1 thiamine for 2 days at 30°C and examined by differential interference contrast microscopy. (C) TYH1 (nmt1-nak1) cells expressing the indicated proteins were grown on minimal medium plates containing 100 µg ml–1 thiamine for 5 days at 30°C.

View larger version (93K): [in a new window]   Fig. 7. Hob1 and Wsp1 cooperate with Nak1 to mediate cell growth and polarity. (A) SPU (wild type), RKO3B-4U (hob1), TYH1 (nmt1-nak1) and RK6B (hob1 nmt1-nak1) strains were grown on minimal medium with 100 µg ml–1 thiamine and with or without 0.5 M KCl at the indicated temperature (30°C or 35°C) for 3-5 days. (B,C) Wild-type (SPU), wsp1 (WSP3-12B) and hob1 (RKO3B-4U) cells expressing HA alone (control) or Nak1-HA from nmt1 promoter expression plasmids were grown on minimal medium for 3 days at 30°C and examined (B) or assayed for growth (C). (D) SPU (wild type), WSP3-12B (wsp1), TYH1 (nmt1-nak1) and NW5D (wsp1 nmt1-nak1) strains were grown on minimal medium with 100 µg ml–1 thiamine at 30°C for 3 days.

View larger version (81K): [in a new window]   Fig. 8. Hob1 and Wsp1 cooperate to mediate cell growth, division and polarity. (A) RNW2D (nmt1-wsp1 hob1) cells were grown to mid-log phase in minimal medium in the absence and presence of 100 µg ml–1 thiamaine and stained with calcofluor to observe cell septae. (B) Wild-type (RL143) cells co-expressing Wsp1-GFP and the myc epitope alone (control, left), myc-Hob1 (middle) or Hob11-281 (right) were grown in minimal medium and viewed for cell morphology and Wsp1-GFP localization.

View larger version (56K): [in a new window]   Fig. 9. Hob1 overexpression or nak1 repression inhibits Wsp1-mediated F-actin formation in vitro. (A) NTA-agarose beads were either coated with Wsp1-HAHis6 by incubation in extracts from yeast expressing this protein or incubated in control yeast lysates (control). They were then incubated with fluorescent Alexa-568/actin and wild-type (SPU) cell extract (WT), wild-type extract in the presence of 10 µM LatA (+LatA) or XB-200 buffer alone (no extract). The ability of the different extracts to stimulate Wsp1-mediated F-actin formation was observed by the presence of fluorescent F-actin halos around the beads. A proportion of the NTA-agarose beads were boiled and immunoblotted with the anti-HA (12CA5) antibody to detect Wsp1-HAHis6 bound to the beads. (B) NTA-agarose beads were similarly incubated in control extracts or extracts from yeast expressing Wsp1-HAHis6 (Wsp1), Wsp1-HAHis6 and myc-Hob1 (+Hob1), or Wsp1-HAHis6 and myc-Hob11-281 (+Hob11-281), and were assayed for the formation of F-actin halos in Alexa-568/actin in the presence of wild-type extract. The presence of Wsp1-HAHis6 (shown on the right) and myc-Hob1 or myc-Hob11-281 (not shown) were confirmed by western blots. (C) Wsp1-HAHis6-coated beads were assayed for F-actin formation in the presence of cell extracts derived from nak1-repressed (TYH1) cells containing a control vector (–Nak1) or expressing either Nak1 (+Nak1) or Nak1T171A (+Nak1T171A) grown in the presence of 100 µg ml–1 thiamine. No F-actin formation was observed in beads coated using control extracts (derived from the wild-type SPU strain, not shown).

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