The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Abstract

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

The flagellar length control system: exploring the physical biology of organelle size

“…Thus, saturable binding of tubulin or other precursors still allows a unique stable steady-state length. We have shown using numerical simulations that the introduction of saturable tubulin binding into the model yields stable length control with regeneration curves similar to those seen in experiments, and with the ability to account, at least qualitatively, for the regeneration behavior of mutants in an IFT associated protein, Crescerin, that is predicted to contain tubulin binding sites (Perlaza et al 2022a).…”

“…The existence of mutations that affect flagellar length is also sometimes taken to indicate the presence of a control system, on the grounds that such genes must encode components of that system. Length mutants of Chlamydomonas fall into two main classes, long-flagella (lf) mutants (Barsel et al 1988;Berman et al 2003, Tam et al 2003, and short flagella (shf) mutants (McVittie 1972, Kuchka and Jarvik 1987, Perlaza et al 2022a, which increase and decrease, respectively, the average flagellar length. The mutants are sometimes interpreted as affecting molecular pathways that control length, and thus their existence is taken as evidence for a length control system.…”

“…Many proteins contribute to flagellar assembly, but tubulin is apparently the most important for length maintenance, as judged by the fact that when both tubulin binding sites on the IFT particle are mutated so that they can no longer bind tubulin, the result is shf (Kubo et al 2016). The fact that the flagella are not zero length suggests that other mechanisms may exist to move tubulin, including other binding sites on IFT particles (Perlaza et al 2022a) or diffusive motion (Craft van de Weghe et al 2020), which will be discussed below in section 3.…”

“…However, when both binding sites were mutated, flagellar length became extremely short (Kubo et al 2016). Flagella were still present, possibly representing either additional tubulin binding sites (Perlaza et al 2022a) or an IFT independent component of tubulin transport. The latter was supported by the ability of mutant tubulins, altered to make them unable to bind to IFT proteins, to enter the flagellum and incorporate into the axoneme (Craft van de Weghe et al 2020).…”

“…The long flagella mutants have received the lion's share of attention from Chlamydomonas geneticists, but from the point of view of understanding the biological mechanism of length control, shf mutants are equally informative. The first short flagella mutant mapped, shf1 (Kuchka and Jarvik 1987) was recently found to represent a mutation in a flagella-localized homolog of the TOG-domain family protein Crescerin (Perlaza et al 2022a). Tumor overexpressed gene (TOG) domains can function both to bind tubulin dimers, and also to promote their polymerization onto growing microtubules (Slep 2009).…”