Determinants of cytoplasmic microtubule depolymerization during ciliogenesis in .

At the core of cilia are microtubules which establish length and assist ciliary assembly and disassembly; however, microtubules outside of the cilium can regulate ciliogenesis. The microtubule cytoskeleton polymerizes and depolymerizes rapidly. These processes have been studied across various organisms with chemical and genetic perturbations.

Determinants of cytoplasmic microtubule depolymerization during ciliogenesis in .

At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which polymerizes and depolymerizes rapidly to assist in cellular processes. These processes have been studied across various organisms with chemical as well as genetic perturbations.

The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement.

MAPK pathways are well-known regulators of the cell cycle, but they have also been found to control ciliary length in a wide variety of organisms and cell types from neurons to mammalian photoreceptors through unknown mechanisms. ERK1/2 is a MAP kinase in human cells that is predominantly phosphorylated by MEK1/2 and dephosphorylated by the phosphatase DUSP6. We have found that the ERK1/2 activator/DUSP6 inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), inhibits ciliary maintenance in and hTERT-RPE1 cells and assembly in These effects involve inhibition of total protein synthesis, microtubule organization, membrane trafficking, and KAP-GFP motor dynamics.

Initial ciliary assembly in requires Arp2/3 complex-dependent endocytosis.

Ciliary assembly, trafficking, and regulation are dependent on microtubules, but the mechanisms of ciliary assembly also require the actin cytoskeleton. Here, we dissect subcellular roles of actin in ciliogenesis by focusing on actin networks nucleated by the Arp2/3 complex in the powerful ciliary model, . We find that the Arp2/3 complex is required for the initial stages of ciliary assembly when protein and membrane are in high demand but cannot yet be supplied from the Golgi complex.

Lithium-induced ciliary lengthening sparks Arp2/3 complex-dependent endocytosis.

Ciliary length is highly regulated, but can be disrupted by lithium, which causes ciliary elongation across cell types and organisms. We used the algal system to investigate the mechanism behind lithium-induced ciliary elongation. Protein synthesis is not required for lengthening, and the target of lithium, GSK3, has substrates that can influence membrane dynamics.

Comparative Phenotyping of Two Commonly Used Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses.

A guide to setting up and managing a lab at a research-intensive institution.

Postdocs who land faculty jobs at research-intensive institutions need to juggle several new large-scale tasks: identifying space and equipment needs for their lab, negotiating the hiring package, outfitting the lab with supplies, building a team, and learning to manage time in ways that can promote productivity and happiness. Here we share tips to help new hires think clearly about each of these tasks.

Finding your unique path in science.

I always found it curious that in science, we value unique, creative thinkers, but we teach scientists to progress in a formulaic manner that rarely takes each person's individual strengths into account. Surprisingly, when we break the mold, we are often rewarded for it. This cycle of learning to survive using conventional wisdom but being rewarded for a unique path outside of it seems to be an unspoken key to success.

Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit.

Kinesin is part of the microtubule-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits, kinesin family member 3A/3B (KIF3A/3B), and kinesin-associated protein 3 (KAP3), is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia.

formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in is the fertilization tubule, a mating structure at the apical cell surface in gametes.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins.

The green alga is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells.

Visualizing Filamentous Actin Using Phalloidin in .

This protocol aims to visualize the filamentous actin network in . We improved fixed-cell labeling conditions using the F-actin probe, phalloidin. We created a -optimized protocol by halving the phalloidin incubation time, electing for optimal fixation conditions, and selecting for a healthy cell population.

Partially Redundant Actin Genes in Chlamydomonas Control Transition Zone Organization and Flagellum-Directed Traffic.

The unicellular green alga Chlamydomonas reinhardtii is a biflagellated cell with two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Here, we probe how actin redundancy contributes to flagellar assembly. Disrupting a single actin allows complete flagellar assembly.

Can microtubule motors use every available track?

Flagellar assembly and function depend on cargo traveling via motors on microtubule doublets. Bertiaux, Mallet et al. (2018.

Erratum to: Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii.

The original version of this chapter was inadvertently published with the incorrect affiliation for Brittany Jack and Dr Prachee Avasthi. This has been corrected and the affiliation details has been updated as follows.

Journal clubs in the time of preprints.

Early-career researchers can learn about peer review by discussing preprints at journal clubs and sending feedback to the authors.

Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii.

Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of vertebrate cells and provide an excellent model to study ciliogenesis. Cilia and flagella are important organelles used for motility and sensing the extracellular environment. Abnormalities in cilia structure or ciliary dysfunction can have devastating consequences ranging from diabetes and obesity to polycystic kidney disease and mental retardation.

Primary Cilia in Cystic Kidney Disease.

Primary cilia are small, antenna-like structures that detect mechanical and chemical cues and transduce extracellular signals. While mammalian primary cilia were first reported in the late 1800s, scientific interest in these sensory organelles has burgeoned since the beginning of the twenty-first century with recognition that primary cilia are essential to human health. Among the most common clinical manifestations of ciliary dysfunction are renal cysts.

Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies.

The unicellular green alga is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle.

Actin is required for IFT regulation in Chlamydomonas reinhardtii.

Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery.

Centrin 2 is required for mouse olfactory ciliary trafficking and development of ependymal cilia planar polarity.

Centrins are ancient calmodulin-related Ca(2+)-binding proteins associated with basal bodies. In lower eukaryotes, Centrin2 (CETN2) is required for basal body replication and positioning, although its function in mammals is undefined. We generated a germline CETN2 knock-out (KO) mouse presenting with syndromic ciliopathy including dysosmia and hydrocephalus.

Ciliary regulation: disassembly takes the spotlight.

The mechanisms that control organelle size are poorly understood. Genetic analysis now shows that a NIMA-related kinase helps to regulate the size of cilia by activating disassembly of the cilium when it gets too long.