Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK‐related kinase

Abstract

Members of the MAPK superfamily are known as key regulators of ciliogenesis. Long flagellar (LF) 4, a MAPK‐related kinase in Chlamydomonas, is the first kinase that was implicated in ciliary assembly and length. However, little is known about its cellular properties, regulation, and molecular functions. LF4 is localized both in the flagella and cell body with enrichment at the 2 basal bodies, shown by super‐resolution microscopy. LF4 is constitutively phosphorylated at T159 at the kinase activation loop and remains at the basal bodies during flagellar assembly. Gene mutations that affect the kinase activity or T159 phosphorylation alter the localization of LF4 at the basal bodies, and the mutants fail to rescue lf4‐3, a null mutant. LF4 does not affect the velocities of intraflagellar transport (IFT). However, LF4 null mutation induces accumulation of IFT proteins in the flagellum and reduces the phosphorylation of the kinesin‐II subunit FLA8/KIF3B, indicating that LF4 negatively regulates IFT entry. Furthermore, LF2, a cell cycle–related kinase, and LF3, a novel protein, are required for LF4 phosphorylation. Our study demonstrates that LF4 is likely a constitutively active kinase that is regulated by LF2 and regulates IFT entry at the basal bodies to control flagellar assembly and length.—Wang, Y., Ren, Y., Pan, J. Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK‐related kinase. FASEB J. 33, 6431–6441 (2019). http://www.fasebj.org

LF4/MOK and a CDK-related kinase regulate the number and length of cilia inTetrahymena

“…Among several metazoan species analyzed, CDKR1 is most similar to DYF-18 (Fig 1A), a known cilia-associated CDK-related kinase of C. elegans , which was proposed to be a homolog of LF2/CCRK CDK kinases [46, 48]. While our phylogenetic analysis does not support either DYF-18 or CDKR1 as orthologs of LF2/CCRK, we note that DYF-18, CDKR1, LF2 and CCRK are all CDK-type kinases that lack the cyclin-binding motif, PSTAIRE, characteristic of the canonical CDKs that regulate the cell cycle (S5C Fig), and are all associated with cilia where they act upstream of RCKs (see below and [40, 43, 47, 48, 64]).…”

“…Importantly, in Chlamydomonas, hypomorphic LF2 alleles confer longer cilia, but a null allele produces variable length (including shorter) cilia and inability to regenerate cilia after deciliation [47, 64, 94]. Loss of either RCKs or LF2/CCRK cause excessive accumulation of IFT materials in cilia, which could create roadblocks that reduce the efficiency of IFT [40-42, 47, 93, 95]. In the multiciliated cells, such as Tetrahymena , the excessively long cilia may deplete factors whose concentration is rate-limiting for ciliogenesis in other parts of the same cell.…”

“…In particular, the anterograde IFT direction was consistently affected by manipulations of LF4A in all assays used. In Chlamydomonas and mammalian cells, deficiencies in LF4/MOK did not change the IFT train velocities [38, 40, 100]. On the other hand, in mammalian cells, a depletion of ICK increased the rate of anterograde IFT and its overexpression reduced the rate of retrograde IFT, respectively [38].…”

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

“…Indeed, it has been demonstrated in HEK293 cells that an increase in cellular cAMP levels and concomitant PKA-dependent protein phosphorylation at centriolar satellites induces ubiquitination and proteolysis of NEK10 by the co-assembled E3 ligase CHIP and promotes cilia resorption (Porpora et al, 2018). This is supported by elegant studies of cilia length control in Chlamydomonas (Cao, Meng et al, 2013, Hendel, Thomson et al, 2018, Hu, Liang et al, 2015, Ishikawa & Marshall, 2017, Liang, Zhu et al, 2018, Ludington, Ishikawa et al, 2015, Luo, Cao et al, 2011, Meng & Pan, 2016, Pan & Snell, 2005, Wang et al, 2019.…”

“…The primary cilium is a dynamic cellular structure that assembles and dissembles in accordance with the cell cycle (Keeling, Tsiokas et al, 2016, Kim & Tsiokas, 2011, Wang, Ren et al, 2019. The interplay between assembly and disassembly determines the length of the primary cilium.…”