Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in \u3ci\u3eTrypanosoma brucei\u3c/i\u3e

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum’s tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2’s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids’ unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei.

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum's tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2's effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally

Trapped cells near motility chamber surface displayed planar ciliary beating.

Movie of an uninduced cell trapped by the optical tweezer approximately 3 μm above the surface of the motility chamber. The cell is constrained by the glass surface. However, the rotation and out-of-plane beating is observable. Movie was recorded using wide-field microscopy with a 60x objective at 45 fps and played back at the same frame rate. (AVI)</p

LC2 knockdown causes mislocalization of kinetoplast and cell division defects.

A. Representative images of uninduced, FLAM3 KD, FLAM3-LC2 KD, and FLAM3-LC2 KD/LC2 OE cells cultures in the culture flask using phase-contrast microscopy 72 hours post-induction when we did not shake (top) and shook (bottom) the flasks. Major clusters of cells are indicated (red arrows). The scale bars represent 10 μm. B. Representative DAPI stained images for classification of cells as having x kinetoplasts (xK) and y nuclei (yN). 1K 1N refers to cells with one kinetoplast normally localized to one nucleus (left). MK MN refers to cells classified as having multiple (M>2) mislocalized (closer to each other) kinetoplasts and nuclei (right), likely resulting from incomplete kinetoplast migration and/or incomplete cytokinesis. The scale bar represents 5 μm and both images in this panel have the same scale. C. Occurrence frequency of one kinetoplast and one nucleus, normally localized within the cell (1K 1N) and the occurrence frequency of the multi-kinetoplast, multi-nucleus (MK MN) classification, as described in panel B., in uninduced and induced (72 hours post-induction) LC2 KD, FLAM3 KD, FLAM3-LC2 KD, FLAM3-LC2 KD/LC2 OE, and WT/LC2 OE cells. N = 101, 192, 122, 111, 72, and 70 total classified cells of each strain, respectively. Other classifications, e.g., 1K 2N, 2K 1N, and 2K 2N, which likely include cells undergoing cell division, account for the percentages not represented. The error bars represent the statistical counting error. ** = p-value D. DIC microscopy image of fixed FLAM3-LC2 KD cells, including a representative amorphous clump of cells with multiple detached cilia (red arrow). (TIF)</p

FLAM3-LC2 KD double knockdown cells show altered ciliary beating.

A high-speed movie of FLAM3-LC2 KD double knockdown cells showing a highly asymmetric forward (tip-to-base) beating with an intermittent reversal in ciliary beat direction—both of the phenomena leading to cell reorientation. The movie was recorded at 200 fps with an exposure time of 250 μs and played back at 45 fps. (AVI)</p

Optically trapped LC2 KD and FLAM3-LC2 KD cells showed upregulated ciliary beat frequency.

A. Schematic of a T. brucei cell (gray) trapped by a low power (~20 mW) optical tweezer (red). B. Brightfield image of an uninduced FLAM3-LC2 KD cell taken near the coverslip, thus enabling direct visualization of the trap location and aligning the cell in the imaging plane. The identified trap location on a trypanosome cell (red arrowhead) is typical of all cells we trapped. Scale bar = 5 μm. C. Typical maximum projection (intensity inverted) of a movie (2 s) showing a trapped FLAM3 KD cell as it rotates about the trap center (red arrowhead). Scale bar = 5 μm. D. Typical power spectral densities (PSD) of a trapped cell with its cilium beating (FLAM3-LC2 KD/LC2 OE cell, orange), a trapped cell with its cilium not beating (FLAM3-LC2 KD/LC2 OE cell stalled at the bottom of the imaging chamber, purple), and background trap noise (gray, about six orders of magnitude smaller than the PSD of the cell with a beating cilium at the beat frequency). Peaks in the PSD represent the characteristic cell rotation rate (blue arrowhead) and ciliary beat frequency (red arrowhead). Each PSD represents the average of 3 spectra (1 second of data, each). E. Ciliary beat frequency, fω, of multiple cell lines obtained from the higher of the two characteristic frequencies from the PSD analysis. The error bars represent the SEM and *** represents p-values 0.05 from two-tailed paired t-tests. N = 25, 86, 75, 85, and 96 for uninduced, LC2 KD, FLAM3 KD, FLAM3-LC2 KD, and FLAM3-LC2 KD/LC2 OE cells, respectively.</p

LC2 KD and WT/LC2 OE do not show cell clustering defects.

Representative images of LC2 KD and WT/LC2 OE cell in the culture flask using phase-contrast microscopy 72 hours post-induction when we did not shake the flasks. We observed no cell clustering. The scale bars represent 10 μm. (EPS)</p

FLAM3 knockdown and TbLC2 overexpression cause shorter <i>T</i>. <i>brucei</i> cilia.

Length distributions of cilia from strains as indicated. The black lines indicate the mean length, and shaded regions around the line indicate the SE of the mean (N = 76, 76, 52, and 101 for WT, FLAM3 KD, FLAM3-LC2 KD, and FLAM3-LC2 KD/LC2 OE cells, respectively). *** represents p-value p-value (EPS)</p

FLAM3-LC2 KD double knockdown cells exhibit ciliary beating locked to a reverse beating over an extensive period showing a longer dwell time in reverse beating mode.

The movie was recorded using a high-speed CMOS camera and a 60x objective at 200 fps with an exposure time of 250 μs and played back at 45 fps. Scale bar = 10 μm. (AVI)</p

Homology modeled structure of TbLC2 using CrLC2 as a template generated by SWISS-MODEL and used and modified under the creative commons license CC BY-SA 4.0.

(PDB)</p