The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.dynein f | flagella C ilia and flagella are highly conserved organelles with roles in cellular movement and signal transduction. In humans, defects in cilia can lead to a number of diseases, such as polycystic kidney disease and primary ciliary dyskinesia (1, 2). The axoneme core of most motile cilia and flagella consists of nine doublet microtubules (DMTs) surrounding two single microtubules (MTs) known as the central pair complex (CPC) (Fig. 1). DMTs are highly periodic with a 96-nm-long unit that repeats along the MT length. They are decorated with two rows of dynein motors, the inner dynein arms (IDAs) and the outer dynein arms (ODAs), which drive MT sliding and axoneme bending (3). Generating the diverse ciliary and flagellar waveforms requires the precise coordination of the activity of thousands of dynein motors within a single organelle (4).The primary signaling pathway known to regulate dynein function in axonemes involves signals traveling from the CPC through radial spokes (RSs) to the IDAs and ODAs (reviewed in 5, 6). Many CPC and RS mutants are paralyzed (7). However, MT sliding can be restored to WT levels in isolated CPC/RS mutant axonemes using protein kinase inhibitors (8, 9). These observations, together with biochemical evidence of phosphorylation of dynein subunits, have implicated the dyneins as a major signaling target of the CPC/RS signaling pathway (10, 11). However, the regulatory mechanisms and physical interactions that participate in signal transduction to the dynein targets are not well understood.Isolated axonemes require only ATP for reactivation, suggesting that direct interactions between the dyneins and their regulators are physically built into the axoneme (4). Therefore, studies that visualize axonemal structures and their connections at high resolution can provide ...

Cryo–electron tomography of Chlamydomonas flagella reveals previously uncharacterized features of the radial spokes, including structural heterogeneity and direct interactions with dyneins and between the spoke heads. A “radial spoke 3 stand-in” occupies what would be the site of a third spoke in organisms with spoke triplets.

Analogous to synaptotagmin 1, a calcium-sensitive regulator of presynaptic vesicle fusion, synaptotagmin 4 needs both of its calcium-binding sites to regulate synaptic plasticity via postsynaptic retrograde signaling.

Seven of 15 different mouse formins localized in diverse patterns to cardiomyocyte sarcomeres. Four were required for proper organization of myofibrils, and two were critical for remodeling and repair of myofibril structure.