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. 2008 Aug 26;18(16):1215-20.
doi: 10.1016/j.cub.2008.07.026. Epub 2008 Aug 7.

Costal2 functions as a kinesin-like protein in the hedgehog signal transduction pathway

Shohreh F Farzan  1 , Manuel Ascano Jr, Stacey K Ogden, Matthieu Sanial, Amira Brigui, Anne Plessis, David J Robbins
Affiliations

Costal2 functions as a kinesin-like protein in the hedgehog signal transduction pathway

Shohreh F Farzan et al. Curr Biol. .

Abstract

The Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues [1]. Although Costal2 (Cos2) is a requisite component of the Hh pathway, its mechanistic role is not well understood. Because of its primary sequence, Cos2 was initially predicted to function as a kinesin-like protein [2]. However, evidence showing that Cos2 function might require kinesin-like properties has been lacking [2-6]. Thus, the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein [7, 8]. Here, we show that Cos2 motility is required for its biological function and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP, and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci). Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function [9]. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed.

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Figures

Figure 1
Figure 1. Hh regulates the subcellular localization of Cos2
(a–a’) Live images of S2 cells expressing cos2-GFP with (+) or without hh. The bulk of Cos2-GFP is found in puncta in the absence of Hh. Hh exposure results in a more diffuse localization of Cos2-GFP. (b–b’”) Live images of S2 cells expressing (b) cos2-RFP only, pseudo-colored green, or (b’–b’”) co-expressing smo-GFP, pseudo-colored red. As with Cos2-GFP, Cos2-RFP is found primarily in puncta. However, co-expression with smo-GFP results in a shift to a more diffuse localization of Cos2-RFP. (c–c’) Indirect immunofluorescent staining of endogenous Cos2 in S2 cells, with (+) or without hh. The majority of Cos2 appears punctate throughout the cytoplasm of the cells. Exposure to Hh leads to an apparent net reduction in fluorescence intensity, decreased localization to puncta and increased diffuse staining of Cos2. (d–d”) Cos2-GFP puncta partially co-localize with Rab5. Approximately 30% of the total Cos2-GFP fluorescence co-localizes with Rab5, a marker of early endosomal vesicles, delineating one pool of membrane vesicle associated Cos2. Inset in lower left-hand corner shows an enlarged area of Cos2 and Rab5 co-localization.
Figure 2
Figure 2. Cos2 puncta are motile
S2 cells expressing cos2-GFP (a), klp10A-GFP (b), a known kinesin family member, or cos2ΔMotor-GFP (c), a GFP-fused truncated form of Cos2 that lacks its putative motor domain. The large box in each panel indicates the enlarged area depicted in the corresponding time series in (a’), (b’) and (c’). The smaller box indicates the origin of a single puncta that is tracked, over time, with arrows in (a’), (b’) and (c’). (a’, b’) Time-lapse images showing a Cos2-GFP puncta (a’) and a Klp10A-GFP puncta (b’) both moving away from their original locations (small boxes). (c’) Time-lapse images showing a Cos2ΔMotor-GFP puncta that remains essentially immobile over the same time period as in a’.
Figure 3
Figure 3. Cos2 motility requires microtubules and ATPase activity
The large box in panels (a) and (b) indicates the enlarged area depicted in the corresponding time series (a’ and b’). The smaller boxes indicate the origin of a single puncta that is tracked, over time. (a– a’) S2 cells expressing cos2-GFP were ATP depleted by treatment with 5 mM sodium azide and 1 mM 2-deoxyglucose. Time-lapse images were taken 30 minutes after ATP depletion. The vast majority of Cos2-GFP puncta were immobile, relative to the vehicle treated S2 cells, which exhibited similar motility to untreated wt Cos2-GFP (Figure S3). (b– b’) Cos2 movement is recovered after ATP levels are restored, verifying the reversibility of the ATP depletion. The same cell as in (a) was reimaged after the ATP depletion solution was removed and the cell was allowed to recover for 30 minutes in fresh media. The movement of a single puncta (indicated by arrows) is tracked over a short time course. (c) Cos2 appears to localize along MT tracks. cos2-RFP was expressed in a S2 cell line stably expressing GFP-α-tubulin and imaged in live cells. The large box in the merge panel indicates the enlarged area depicted in the 2x merge panel. Individual Cos2-RFP puncta aligning with MTs are marked by arrows in the rightmost panel. (d) Live images of S2 cells co-transfected with plasmids expressing cos2-GFP and mCherry-tubulin, then treated with nocodazole. Images show a single cell over the course of 6 hours and show the correlation between dissociation of Cos2-GFP puncta and MT destabilization induced by nocodazole treatment. A more extensive time course of this nocodazole treated cell, showing Cos2-GFP and mCherry-tubulin in separate channels, is shown in Fig. S5a. (e) Live images of S2 cells expressing cos2-GFP and mCherry-tubulin, treated with 5 µM cytochalasin for 18 hours. Cytochalasin disrupts the actin cytoskeleton, allowing the formation of long MT-enriched projections, along which Cos2-GFP puncta appear to co-localize and move. The puncta indicated by the arrow traveled approximately 8 µm along the visible MT projection. (f–f’) Live images of S2 cells expressing cos2-S182N-GFP. (f) Cos2-S182N-GFP shows a highly punctate staining pattern in a subset of cells, similar to wt Cos2-GFP. The large box in panel (f) indicates the enlarged area depicted in the corresponding time series (f’) and the smaller box indicates the origin of a single puncta that is tracked, over time. (f’) Cos2-S182N-GFP puncta tracked over time do not exhibit significant motility (see also Fig. S3b).
Figure 4
Figure 4. Cos2 recruits and moves other components of the Hh signaling pathway
GFP-fu-tail (a, b) and GFP-ci-CORD (c, d) were expressed in S2 cells in the presence or absence of exogenous wt cos2. (a and b) Live images of S2 cells expressing GFP-fu-tail. GFP-Fu-tail localizes in a diffuse manner when expressed alone (a), but co-expression with wt cos2 dramatically relocalizes GFP-Fu-tail into puncta (b), which move with a similar velocity (b’) as Cos2 puncta. (c and d) Live images of S2 cells expressing GFP-ci-CORD. As previously observed in Cl8 cells [11], GFP-Ci-CORD is mostly nuclear (c). The expression of exogenous cos2 leads to a marked relocalization of Ci-CORD to cytoplasmic puncta (d), which were also shown to be motile (d’). (e) Cos2-RFP co-localizes with Smo-GFP in a subset of motile puncta. Live images of S2 cells expressing cos2-RFP and smo-GFP show that a subset of puncta co-localize and are motile (e’). Corresponding single fluorescence images of Smo-GFP and Cos2-RFP are shown in Fig. S6c. The large box in panels (b), (d), and (e) indicates the enlarged area depicted in the corresponding time series (b’, d’, and e’). The smaller boxes indicate the origin of a single particle (arrow) that is tracked, over time.
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References

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