Recording and Analyzing IFT in Chlamydomonas Flagella

Abstract

The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.

A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

“…Cells were applied to cleaned microscope slides with a drop of M medium or M medium+the appropriate concentration of BFA prepared in 2.5% low EEO agar. Cells were recorded at 1 min intervals for 120 min with low illumination and recorded with a Zeiss WL microscope with a UV filter and 40×/0.75 or 63×/1.4 phase contrast lenses [12], [79].…”

Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians

“…Videos were captured at 180 frames per second using a Flare 4M180CCL digital camera (IO Industries, www.ioindustries.com) and were edited with Virtualdub (www.virtualdub.org) to adjust for brightness/contrast, crop the specific region of interest and decompile videos for the isolation of individual jpeg images. In order to determine ciliary beat frequency, a small rectangular region of interest perpendicular to the lateral body wall was selected from each video, and this was used to generate kymographs using ImageJ software (NIH) and methods as previously described (Dentler et al, 2009). From the kymograph images, the number of cycles that individual motile cilia are able to complete during the selected time frame of the video segment were counted, and the beat frequency was calculated for that particular specimen.…”

In vivo Imaging of IFT in Chlamydomonas Flagella

“…This can be done by "gluing" cells to coverslips pretreated with poly-L-lysine (e.g., 0.1%, w/v, in water), embedding cells in low melting agarose (e.g., 1% seagate low melting agarose; 1-2% EEO agarose), or treatment with paralyzing agents such as 20 mM LiCl (Dentler, Vanderwaal, & Porter, 2009;Diener, 2009). reinhardtii cells propel themselves by means of two motile flagella.…”

“…However, IFT has been also observed using a standard halogen lamp (Dentler et al, 2009). Microscope equipped with prisms and analyzer for DIC illumination.…”

“…15.1E and Fig. Algorithms to enhance contrast and visibility of moving particles have been described in Dentler et al (2009), Iomini et al (2001, and Pazour, Wilkerson, and Witman (1998). Kymograms allow to quantitatively determine IFT parameters such as particle velocity, frequency, and size.…”