Forces and Disease: Electrostatic force differences caused by mutations in kinesin motor domains can distinguish between disease-causing and non-disease-causing mutations

The ability to predict if a given mutation is disease-causing or not has enormous potential to impact human health. Typically, these predictions are made by assessing the effects of mutation on macromolecular stability and amino acid conservation. Here we report a novel feature: the electrostatic component of the force acting between a kinesin motor domain and tubulin. We demonstrate that changes in the electrostatic component of the binding force are able to discriminate between disease-causing and non-disease-causing mutations found in human kinesin motor domains using the receiver operating characteristic (ROC). Because diseases may originate from multiple effects not related to kinesin-microtubule binding, the prediction rate of 0.843 area under the ROC plot due to the change in magnitude of the electrostatic force alone is remarkable. These results reflect the dependence of kinesin’s function on motility along the microtubule, which suggests a precise balance of microtubule binding forces is required

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

Optimization of Modular, Long-Range, Ultra-Fast Optical Tweezers With Fluorescence Capabilities for Single-Molecule and Single-Cell Based Biophysical Measurements

An Optical tweezer is a tightly focused laser beam that applies and senses precise and localized optical force to a dielectric microsphere and offers a unique and effective tool for manipulating the single cell or cell components, including nucleotides and dynein motor proteins. Here, I used highly stabilized optomechanical components and ultra-sensitive detection modules to significantly improve the measurement capabilities over a wide range of temporal and spatial scales. I combined the optical tweezer-based force spectroscopy technique with fluorescence microscopy to develop an integrated high-resolution force-fluorescence system capable of measuring displacements at sub-nanometer, forces at sub-piconewton over a temporal range of milliseconds to 10s of seconds. I developed a flow cell-based high throughput DNA tether assay to probe the correlation between the structure, dynamics, and functionality of Thiamine pyrophosphate binding RNA Riboswitch. I used this dynamic single-molecule assay to analyze the metabolite-dependent dynamic switching behavior of the riboswitch. Furthermore, I used a high throughput single-cell trapping assay in combination with high-speed video microscopy to characterize the dynein regulatory role of light chain-2 in dynein-based flagellar motility in the Trypanosoma brucei, a parasitic protozoan. Finally, I used the fluorescence capability of the system to visualize the fluorescently labeled microtubules and study the molecular interactions between the dynein motor protein and microtubule in either the single-molecule unbinding assay performed using mouse cytoplasmic dynein MTBD or bulk protein gliding assay performed using the Chlamydomonas or Trypanosome flagellar dyneins. I expect understanding molecular interactions provides a platform for their use as novel therapeutic targets

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

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

Template-free model of the full-length TbLC2 built using AI-based AlphaFold modeling.

(PDB)</p

Stable assembly of outer arm dynein into the axoneme does not require TbLC2.

Transmission electron microscopy (TEM) images of wild-type (WT, left), FLAM3-LC2 KD (middle), and FLAM3-LC2 KD/LC2 OE (right) cells show the axoneme (canonical 9+2 microtubule arrangement) and paraflagellar rod (yellow arrowheads). The outer arm (red arrowheads) and inner arm (blue arrowheads) dyneins were intact in all three cell lines. The scale bar is 100 nm, and all micrographs have the same magnification.</p

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

Characteristic beating mode dwell times.

Dwell time is the fit parameter ± the standard error of the fit, obtained by fitting exponential functions to the cumulative probability distributions of the dwell times (Fig 7F).</p

Curvature during the highly asymmetric reversed base-to-tip beating in FLAM3/LC2 double knockdown cell.

The curvature normalized to contour length (κ/L) plotted as a function of normalized arc length (s/L) along the cilium for a typical highly asymmetric base-to-tip wave propagation in FLAM3-LC2 KD cells. s/L of zero represents the ciliary base, and the colors (red to magenta) represent time progression during the propagation of a ciliary bend in a single ciliary beat period. The t = 0 beat profile (red) represents ciliary bend initiation towards the base (left side), followed by the successive profiles representing the propagation of the bend towards the tip (right side). (EPS)</p