Heterogeneity in cilia patterning enables multiple flow functions in Paramecium

Key results

1. ShownFeeding depends on domain D1 (dense cilia anterior to the oral apparatus, OA). Cells lacking D1 cilia ingest essentially no beads. Cells keeping only D1 and OA cilia feed like treated cells with minor, uniform cilia loss. OA cilia alone do not support uptake.

   Intracellular/background fluorescence ratio after 10-15 min with 1 µm beads: control 1.32 ± 0.10, Uniform 1.21 ± 0.11, D1+OA 1.18 ± 0.11 (ns vs Uniform), D2+OA 0.99 ± 0.03 (significantly below Uniform, like fully deciliated or OA-only cells). Untreated, unvortexed cells: 1.49 ± 0.14. Mann-Whitney U with Holm correction; 6 to 22 cells per condition. · DISTINCT CELLULAR REGIONS CARRY OUT SPECIFIC…; THE CILIARY ARRAY GENERATES STEREOTYPED FLOWS…; e. Feeding analysis; f. Statistical analysis

2. ShownSwimming depends on domain D2 (the sparser cilia over the rest of the cell). Cells keeping only D1 and OA cilia barely move. Cells lacking D1 show no significant loss of speed (vs uniformly ciliated treated cells) and no change in gait time fractions or transitions (vs untreated cells).

   Speed: control 0.41 ± 0.04 mm/s, Uniform 0.43 ± 0.25, D2+OA 0.30 ± 0.06 (ns vs Uniform), D1+OA 0.09 ± 0.07 (significantly slower); OA-only cells immotile. D2+OA cells (n=11) spent 91 ± 6% of time in forward swimming vs 88 ± 8% for untreated cells (n=59). Transition probabilities from forward swimming to reverse/reorient: 0.7 ± 0.1/0.3 ± 0.1 vs 0.7 ± 0.2/0.3 ± 0.2, none significant. · DISTINCT CELLULAR REGIONS CARRY OUT SPECIFIC…; THE CILIARY ARRAY GENERATES STEREOTYPED FLOWS…; f. Statistical analysis; Tables

3. ShownThe cortex divides into a dense domain (D1) anterior and right of the oral apparatus, where basal bodies sit in ciliated pairs, and a sparser domain (D2) over the rest of the ventral surface and all of the dorsal surface. Cilia density is about 1.4-fold higher in D1.

   Expansion microscopy: intra-row spacing 2.28 ± 0.22 µm between basal body pairs in D1 vs 1.89 ± 0.17 µm between basal bodies in D2 (n=10 cells); inter-row 2.28 ± 0.16 vs 1.93 ± 0.25 µm (n=27/28 cells). Because both basal bodies of each D1 pair are ciliated and fewer D2 basal bodies are, cilia density is 1.4-fold higher in D1. Areas: D1 16.1 ± 2.8%, D2 80.4 ± 3.0%, OA 3.5 ± 1.5% (n=12). · CILIA ARRAYS WITH DISTINCT DENSITY AND…; a. Measurement of cell shape and cilia array…; Figure legends

4. ShownD1 cilia beat about twice as fast as D2 cilia but have the same metachronal wavelength, so waves travel roughly twice as fast in D1. In both domains waves head anteriorly at ∼30° to the anterior-posterior (A-P) axis, while stroke direction follows the local striated fibers.

   High-speed DIC (1000 fps) of immobilized cells, n=15: beat frequency 35.4 ± 3.0 Hz in D1 vs 15.7 ± 2.5 Hz in D2; wavelength 9.1 ± 1.0 µm vs 9.1 ± 0.6 µm; wave speed 320.6 ± 39.9 vs 143.9 ± 26.7 µm/s. Waves are dexioplectic in D1 and mixed dexioplectic/antiplectic in D2. Paroral membrane cilia: 30.2 ± 2.1 Hz, stroke at 80.6 ± 7.7° to the A-P axis. · INDIVIDUAL CILIA DYNAMICS AND COLLECTIVE…; b. Analysis of ciliary beat direction; c. Quantification of metachronal wave properties; Figure legends

5. SuggestedParticle image velocimetry (PIV) shows two counter-rotating vortices and stronger ventral flow aimed at the oral apparatus. Removing D1 erases the mouth-directed anterior flow. Removing D2 keeps the ventral flow but leaves negligible dorsal flow.

   Untreated immobilized cells (n=4): ∼650 µm/s ventral vs ∼250 µm/s dorsal near the cell, decaying to 100 µm/s at 200 µm. D2+OA cells (n=5) kept posteriorly shifted vortices, and their normalized radial profiles matched uniformly ciliated treated cells (n=3). D1+OA cells (n=11) matched those cells (n=3) ventrally but had negligible dorsal velocity at every distance. · THE CILIARY ARRAY GENERATES STEREOTYPED FLOWS…; DISTINCT CELLULAR REGIONS CARRY OUT SPECIFIC…; g. Analysis of flow fields; Figure legends