The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

Abstract

Funding informationTIFR, DAE, Government of India; DAE-SRC fellowship; UGC fellowship.The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and α-tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin-treated microtubule and LC-ESI-MS/MS characterization of the tail-fragment-associated tubulin identified an association between the tail domains and α-tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue-cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α-tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a nonparticulate form requiring kinesin-2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia. K E Y W O R D Santenna, cilia, Drosophila, Kif3A, Kif3B, kinesin-like-protein 64D, kinesin-like-protein 68D, mouse

Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

“…Taken together, our results argue that the C-terminal tail of KLP3B augments processivity, possibly by tethering the motor to the microtubule [52], which prevents premature dissociation. Attaching the motor at its C-terminus to beads in previous optical tweezers studies therefore likely curtailed the processivity of the kinesin-2 motor.…”

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

“…Recent studies have, however, stated that the motor subunits could independently interact with soluble proteins through the tail domain (Girotra et al, 2016; Sadananda et al, 2012). Here, we showed that the Kinesin-2α tail could also bind to a membrane-associated protein, Rab4.…”

Cilium structure, assembly, and disassembly regulated by the cytoskeleton

“…For example, kinesin-2, which binds the α-tubulin C-terminus in vitro , has different binding affinities to tubulin isotype mixtures specific to different tissues. Overexpression of the kinesin-2 tail domain causes ciliogenesis defects [ 50 ], indicating that limiting motor protein affinity on the basis of tubulin isotype may function in regulating ciliary stability. Intriguingly, detailed correlative live cell imaging and EM in Chlamydomonas flagella demonstrated that anterograde IFT occupies B tubules, while retrograde IFT prefers A tubules of the same axoneme doublet; thus, a single doublet bears bidirectional cargo transport [ 51 ] ( Figure 2 ).…”