PDF(Pubmed)
Abstract：
The heterodimeric kinesin-2 motor (KIF3A/KIF3B with accessory protein KAP3) drives intraflagellar transport, essential for ciliogenesis and ciliary function. Three point mutations in the KIF3B subunit have recently been linked to disease in humans (E250Q and L523P) and Bengal cats (A334T) (Cogné et al., Am. J. Hum. Genet., 2020, 106, 893-904). Patients display retinal atrophy and, in some cases, other ciliopathy phenotypes. However, the molecular mechanism leading to disease is currently unknown. Here, we used Kif3a -/- ;Kif3b -/- (knockout) 3T3 cells, which cannot make cilia, to characterize these mutations. While reexpression of KIF3B(E250Q) and KIF3B(L523P) did not rescue ciliogenesis, reexpression of wildtype or KIF3B(A334T) restored ciliogenesis to wildtype levels. Fluorescent tagging revealed that the E250Q mutant decorated microtubules and thus is a rigor mutation. The L523P mutation, in the alpha-helical stalk domain, surprisingly did not affect formation of the KIF3A/KIF3B/KAP3 complex but instead impaired motility along microtubules. Lastly, expression of the A334T motor was reduced in comparison to all other motors, and this motor displayed an impaired ability to disperse the Golgi complex when artificially linked to this high-load cargo. In summary, this work uses cell-based assays to elucidate the molecular effects of disease-causing mutations in the KIF3B subunit on the kinesin-2 holoenzyme.
摘要：
异二聚体驱动蛋白2运动（KIF3A/KIF3B与辅助蛋白KAP3）驱动步行内运输，对于纤毛发生和纤毛功能至关重要。KIF3B亚基中的三个点突变最近与人类（E250Q和L523P）和孟加拉猫（A334T）的疾病有关（Cogné等人。,Am.J.哼.Genet.,2020、106、893-904）。患者表现出视网膜萎缩，在某些情况下,其他纤毛病表型。然而,导致疾病的分子机制目前尚不清楚。这里,我们使用Kif3a-/-;Kif3b-/-（敲除）3T3细胞，不能制造纤毛，来表征这些突变。虽然KIF3B（E250Q）和KIF3B（L523P）的再表达不能挽救纤毛发生，野生型或KIF3B(A334T)的再表达将纤毛发生恢复至野生型水平。荧光标记显示E250Q突变体修饰了微管，因此是严格的突变。L523P突变,在阿尔法螺旋茎域中，令人惊讶的是，它没有影响KIF3A/KIF3B/KAP3复合物的形成，而是沿着微管的运动性受损。最后,与所有其他电机相比，A334T电机的表达降低了，当人工连接到这种高负荷货物时，这种马达显示出分散高尔基复合体的能力受损。总之,这项工作使用基于细胞的测定法来阐明KIF3B亚基中致病突变对驱动蛋白2全酶的分子效应.
