Lea M. Alford

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.

An Extended Anaphase Signaling Pathway for Mad2p Includes Microtubule Organizing Center Proteins and Multiple Motor-dependent Transitions

An Extended Anaphase Signaling Pathway for Mad2p Includes Microtubule Organizing Center Proteins and Multiple Motor-dependent TransitionsSignaling pathways within the mitotic mechanism temporally orchestrate spindle assembly withchromosome capture and alignment, and then coordinate initiation of chromosome segregationwith spindle breakdown and cytokinesis for reproductive success. Kinetochore localized Mad2pacts in the spindle assembly checkpoint pathway during prophase and prometaphase to monitorbipolar attachment of chromosomes to spindle microtubules as well as proper tension atkinetochores. Once established, Mad2p is not degraded, but instead transits to spindle polespreceding the metaphase/anaphase transition in human and yeast cells. Whether conservedrelocalization of Mad2p to poles is a final step in the spindle assembly checkpoint pathway orwhether the post-metaphase transition allows Mad2p to cooperate in anaphase events leading tomitotic exit has been unknown. We examined post-metaphase localization of Mad2p in fissionyeast. Our observations indicate an extended signaling pathway for Mad2p that includeskinetochore to bipolar localization at spindle poles, then additional transitions from bipolar tounipolar to equatorial. We determined that Mad2p associates with the microtubule organizingcenter complex through direct binding to Alp4p and that microtubule motor proteins Kinesin-14Pkl1 and Dynein contribute to Mad2p anaphase transitions. At anaphase B onset, bipolar tounipolar transitions of both Mad2p and the septation intitiation network (SIN) kinase Cdc7 areobserved. We determined that Mad2p and Cdc7p transitions monitor different events inanaphase, but that neither are required for anaphase B initiation. Our findings indicate thataltered Mad2p anaphase spindle localizations can reflect changes in spindle function duringmitotic exit that could contribute to fidelity in anaphase events.

Cell polarity emerges at first cleavage in sea urchin embryos.

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G(M1), integrins, and the aPKC-PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical-basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G(M1), integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.

The Ciliary Inner Dynein Arm, I1 dynein, is assembled in the Cytoplasm and Transported by IFT before Axonemal Docking

To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1‐deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild‐type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin‐2 mutant, fla10‐1 revealed that transport of I1 dynein is dependent on kinesin‐2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.

The Chlamydomonas mutant pf27 reveals novel features of ciliary radial spoke assembly.

To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. During ciliary regeneration in pf27, radial spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild‐type Chlamydomonas, radial spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild‐type and pf27 reveals rescue of radial spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of radial spokes occurred independently of the established proximal radial spokes in pf27 axonemes in dikaryons. These results reveal that 20S radial spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for radial spoke–IFT interaction.

FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. Little is known about the assembly and functions of the three individual radial spokes, RS1, RS2, and RS3. In Tetrahymena, a conserved ciliary protein, FAP206, docks RS2 and dynein c to the doublet microtubule.

Polar expansion during cytokinesis

Vesicle trafficking and new membrane addition at the cleavage furrow have been extensively documented. However, less clear is the old idea that expansion at the cell poles occurs during cytokinesis. We find that new membrane is added to the cell poles during anaphase, causing the plasma membrane to expand coincident with the constriction of the contractile ring and may provide a pushing force for membrane ingression at the furrow. This membrane addition occurs earlier during mitosis than membrane addition at the furrow and is dependent on actin and astral microtubules. The membrane that is added at the polar regions is compositionally distinct from the original cell membrane in that it is devoid of GM1, a component of lipid rafts. These findings suggest that the growth of the plasma membrane at the cell poles during cell division is not due to stretching as previously thought, but due to the addition of compositionally unique new membrane.

The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

We developed quantitative assays to test the hypothesis that the N‐DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed “reactivated cell models.” ATP‐induced reactivation of wild‐type cells resulted in the forward swimming of ∼90% of cell models. ATP‐induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark‐field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild‐type reactivated cell models remained intact. The sup‐pf4 and drc3 mutants, unlike other drc mutants, retain most of the N‐DRC linker that interconnects outer doublet microtubules. Reactivated sup‐pf4 and drc3 cell models displayed nearly wild‐type levels of forward motility. Thus, the N‐DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP‐induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N‐DRC indicate B‐tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N‐DRC function.

Kinesin-13 regulates the quantity and quality of tubulin inside cilia

Kinesin-13, a microtubule-end depolymerase, has been shown to affect the length of cilia, but its ciliary function is unclear. In Tetrahymena thermophila, kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and affects the ciliary dynein-dependent motility.

Analysis of Axonemal Assembly During Ciliary Regeneration in Chlamydomonas

Chlamydomonas reinhardtii is an outstanding model genetic organism for study of assembly of cilia. Here, methods are described for synchronization of ciliary regeneration in Chlamydomonas to analyze the sequence in which ciliary proteins assemble. In addition, the methods described allow analysis of the mechanisms involved in regulation of ciliary length, the proteins required for ciliary assembly, and the temporal expression of genes encoding ciliary proteins. Ultimately, these methods can contribute to discovery of conserved genes that when defective lead to abnormal ciliary assembly and human disease.