Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

Abstract

In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles.cilia length ͉ dyf-5 ͉ intraflagellar transport C ilia are present on almost every vertebrate cell and have important functions in motility or sensation. Within cilia, structural components and signaling molecules are transported by a specialized system, called intraflagellar transport (IFT) (1-5). Transport from the base of the cilia to the tip (anterograde) is mediated by kinesin-2 motor complexes, whereas dynein motor complexes mediate transport back to the base (retrograde). The nematode Caenorhabditis elegans has 60 ciliated neurons, including eight pairs of amphid neurons exposed to the environment (6). The cilia of these neurons can be divided into a middle segment with nine doublet microtubules and a distal segment with nine singlet microtubules (7). In the middle segments, two distinct kinesin-2 motor complexes mediate anterograde transport, heterotrimeric kinesin II, encoded by klp-11, klp-20, and kap-1 and homodimeric OSM-3 kinesin (8). In the distal segments, transport is mediated by only OSM-3 (8). Live imaging of the movement of these kinesins suggests that kinesin II alone moves at 0.5 m/s, and OSM-3 alone moves at 1.3 m/s, whereas the two motor complexes together move at 0.7 m/s (8). Recently, Pan et al. (9) have shown that these in vivo transport rates can be reconstituted in vitro by using purified kinesin II and OSM-3 motors. Thus far, two proteins have been identified that are required to stabilize IFT complexes transported by both kinesin II and OSM-3, BBS-7 and BBS-8 (10). However, it remains unclear how kinesin II is restricted to the cilia middle segments, whereas OSM-3 is allowed to enter the distal segments and what the functional significance is of this compartmentalization.Recently, Evans e...

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

“…Mutations in rhodopsin or protein transport machinery of the cilia (e.g., Kif3a or IFT mutants) cause accumulation of rhodopsin in the photoreceptor cell body and result in photoreceptor cell death. Absence of Dyf-5, a nematode homolog of Mak, affects the motility of kinesin motors and IFT particles in the cilia (14). Similarly, we identified aberrant accumulations of IFT and kinesin in the Mak-KO photoreceptor cilia.…”

“…In the distal segment of the amphid channel cilia in dyf-5 animals, singlet microtubules were observed (14). We observed singlet microtubules in outer-segment axonemes, which are positioned near the disk clefts, in both wild-type and Mak-null retinas (Fig.…”

“…LF4 encodes a protein highly similar to mammalian male germ cell-associated kinase (Mak) and intestinal cell kinase (ICK) (13). Loss of function of the LF4 homologs Caenorhabditis elegans dye-filling defective 5 (Dyf-5) and Leishmania Mexicana LmxMPK9 also causes slightly elongated cilia or flagella (14,15). However, molecular regulatory mechanisms controlling ciliary length remain unknown.…”

Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling

“…Similarly, depletion of Ccrk in cultured cells results in elongated cilia (25). In addition, a forward genetic screen of C. elegans identified dyf-5 kinase, an ortholog of LF4, as a regulator of ciliary length and function (7). In humans, loss of MAK, a homolog of LF4 and paralog of ICK, results in elongated cilia in photoreceptor cells, causing retinitis pigmentosa (5,26,27).…”

“…It was shown that Long Flagellar 4 (LF4), the homolog of RCK kinases in Chlamydomonas, is localized in flagella, and null mutants of LF4 possess flagella that are twice as long as those in wild types (6). Furthermore, in Caenorhabditis elegans, disruption of the RCK homolog dye-filling defective 5 (dyf-5) results in the elongation of neuronal cilia, whereas overexpression of DYF-5 leads to the shortening of cilia (7). Although the physiological functions and downstream targets of RCK family kinases are not well understood in higher organisms, these studies suggest that the role of RCK family kinases in controlling ciliary length may be well conserved from single-celled organisms to mammals.…”