Imaging Intraflagellar Transport in Mammalian Primary Cilia

Abstract

The primary cilium is a specialized organelle that projects from the surface of many cell types. Unlike its motile counterpart it cannot beat but does transduce extracellular stimuli into intracellular signals and acts as a specialized subcellular compartment. The cilium is built and maintained by the transport of proteins and other biomolecules into and out of this compartment. The trafficking machinery for the cilium is referred to as IFT or intraflagellar transport. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella. Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations. Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling.

Intraflagellar transport proteins cycle between the flagellum and its base

“…It unravelled the fastest IFT speed reported so far, especially for retrograde transport (Table 3). By comparing IFT data from multiple systems, we noticed a tendency for faster IFT in longer flagella (Table 3), a fact that is supported by the progressive increase in IFT (Engel et al, 2009;Dentler, 2005) during flagellar regeneration in Chlamydomonas or by faster anterograde IFT in IMCD cells incubated with compounds that trigger elongation of the primary cilium (Besschetnova et al, 2009). It should be noted that the predicted elapsed time for a complete IFT cycle is quite similar in all systems reported so far (10-13 s), with the noticeable exception of primary cilia in IMCD cells (Table 3).…”

“…However, these observations are limited to few cell types (Follit et al, 2006;Absalon et al, 2008;Tran et al, 2008) and detailed investigation is only available for Chlamydomonas (Dentler, 2005) and for different types of ciliated neurons in C. elegans where unexpected differences have been reported (Snow et al, 2004;Morsci and Barr, 2011). Imaging IFT in mammals has been challenging and data have only been reported for the primary cilium of LLC-PK1 and IMCD cultured cells (Follit et al, 2006;Besschetnova et al, 2009). These studies revealed quite significant differences in IFT rates and in the nature of IFT anterograde motors and highlight the importance for further investigation in distinct cellular contexts.…”

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis

“…It has also been previously proposed that cilia length can be regulated through the modulation of retrograde and anterograde cargo trafficking along the axoneme (44,51). We have shown that CDK5 inhibition with either R-roscovitine or siRNA did not affect the velocities of the cargo trafficking, suggesting another mechanism must account for shortening of primary cilia length.…”

Loss of the Ciliary Kinase Nek8 Causes Left-Right Asymmetry Defects

“…69 GCaMP3-positive cells were grown in MatTek chambers (35-mm petri dishes with a bottom coverslip insert) to 40%-50% confluency before serum starvation (0.5% FBS) (MatTek, No. 1.5).…”