A simple kinetic model for dynein oscillatory activity

A kinetic model was proposed for dynein, a motor protein, complexed with microtubule fragments. The model explains the experimental observations of oscillatory movements in surprisingly simple axoneme fragments perfused with an ATP solution. This is the first model explaining the oscillatory activity of dynein as determined by a cooperative interaction of two dynein heads in the axoneme. The oscillation shape, frequency, and amplitude obtained for the model are close to the corresponding parameters determined experimentally.     

A comparison of the effects of gentle heating,acetone, and the sulfhydryl reagent bis (4-fluoro-3-nitrophenyl) sulfone on the ATPase activity and pellet height response of tetrahymena cilia

Incubation of glycerol-extracted, Triton X-100 demembranated Tetrahymena cilia with 2–10 vol % acetone caused an enhancement of ATPase activity by 2- to 3- fold, depending on concentration and time of incubation. Axonemal ATPase activity was also increased upon incubation with bis (4-fluoro-3-nitrophenyl) sulfone (FNS). Acetone and FNS enhanced the activity of solubilized 30S dynein, but slightly inhibited that of 14S dynein. Heating at 38°C, incubation with FNS, and incubation with acetone activated axonemal ATPase to the same extent. Subsequent studies of (1) the effect of time of preincubation with a spin-labeled maleimide (SLM) at 25°C as a function of pH on the ATPase activity, (2) the concentration dependence of the inhibition of ATPase activity by N-ethylmaleimide or SLM, (3) the ratio of ATPase activity assayed at 25°C to that assayed at 0°C, and (4) the ratio of ATPase activity at pH 8.6 to that at pH 6.9 did not reveal any difference in the properties of the axonemal ATPase after near maximal enhancement by the heat, acetone, or FNS treatments. It was concluded that enhancement of ATPase activity by gentle heat treatment, by incubation with acetone (or other organic solvents), or by FNS results from a conformation change of 30S dynein. The effect of acetone and of FNS on the pellet height response (a measure of the increase in height of the pellet of cilia precipitated by brief centrifugation in the presence of ATP as compared to the absence of ATP) was also determined. Enhancement of ATPase by these reagents did not lead to a decrease in pellet height response. This observation, in conjunction with other data, indicates that there are at least 3 states of the cross-bridge cycle of dynein arms in cilia.     

IFT54 directly interacts with kinesin‐II and IFT dynein to regulate anterograde intraflagellar transport

The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin‐II, the retrograde motor IFT dynein, and the IFT‐A and ‐B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT‐B protein IFT54 interacts with both kinesin‐II and IFT dynein and regulates anterograde IFT. Deletion of residues 342–356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin‐II and this interaction was strengthened for the IFT54^Δ342–356 mutant in vitro and in vivo. The deletion of residues 261–275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC, and deletion of residues 261–275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.     

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.