Fig 2: Disease-associated mutations in ALMS1 induce reciprocal amplification of the cartwheel and centriole via a positive feedback loop.a Quantification of SAS-6 foci in ALMS1∆ex8 cells arrested at S or G2 phase. S or G2 cells were enriched by treating unsynchronized ALMS1∆ex8 cells with aphidicolin (2 μg/ml) or RO-3306 (9.5 μM), respectively, for 20 hrs. Data are mean ± s.d., with N = 3 independent biological replicates. Source data are provided as a Source Data file. b Summary of all forms of cartwheel amplification seen in G2-arrested ALMS1∆ex8 cells from (a). The mother centriole (dark green), daughter centriole (light green), cartwheel (red), and PCM (yellow) were marked as indicated. Cartwheel amplification in each cell could be classified into one of the 12 configurations using 1×3 matrix representations, with the first number indicating the total number of cartwheels per cell, the second indicating the number of cartwheels associating with engaged centrioles (green doublets), and the third indicating the number of cartwheels associating with disengaged centrioles (green singlets). c, d Time-course analyses of cartwheel dynamics in ALMS1WT (c) or ALMS1∆ex8 (d) cells synchronously arrested in G2 by RO-3306 (9.5 μM). Cartwheel amplification was seen in ALMS1∆ex8 cells (d, left), which was completely suppressed by PLK1 inhibition (200 nM BI-2536) applied 3 h after adding RO-3306 (d, right). Data are mean ± s.d., with N = 3 independent biological replicates. Source data are provided as a Source Data file. e ALMS1∆ex8 cells expressing centrin:GFP and synchronously entering and being arrested at G2 for 6 h by RO-3306 (9.5 μM) were filmed by time-lapse microscopy for an additional 10 hrs. Still images of a G2 cell carrying two pairs of engaged centrioles undergoing disengagement were extracted from the movie. Split arrows indicate centriole disengagement. Box b1 at the 55 min timepoint marks reduplication. f Early phases of cartwheel splitting and shedding that accompany centriole disengagement in NF2-/-; p53-/-; ALMS1∆ex8 cells synchronously arrested at G2 for 6 or 12 h. Daughter centrioles disengaging from mothers were marked by CNTROB labeling (magenta). g Ex-dSTORM analyses of the cartwheel splitting and shedding in ALMS1∆ex8 cells arrested in G2. Top, a pair of ALMS1∆ex8 centrioles initiating cartwheel shedding, accompanying centriole disengagement. Note that the cartwheel had physically split, with the proximal unit partially exiting the lumen. Bottom, a pair of ALMS1∆ex8 centrioles that had shed one cartwheel unit while keeping the other unit in the lumen. h UExM images of the 3-1-2 and 4-0-4 configurations seen in intact ALMS1∆ex8 cells, showing cartwheel shedding and centriole disengagement. Note the location of the cartwheel, some still in the lumen while some tethered to centrioles at the side. i UExM images of the 3-2-1 (i), 4-1-3 (ii) and 4-2-2 (iii) configurations in intact ALMS1∆ex8 cells showing procentriole growth. White arrows indicate newly formed procentrioles (or granddaughter centrioles). Note that CNTROB labeling marked both daughter and granddaughter procentrioles (magenta) in one of the 4-2-2 cells. All DNA was shown in blue (DAPI). All scale bars, 1 μm (white) and 5μm (yellow). j Representative images showing cartwheel shedding in concert with centriole disengagement (top) and procentriole growth at abnormal locations (bottom) in ALMS1∆ex8 cells arrested at G2. Note the abnormal location of the cartwheel or procentriole below the proximal end of the host centriole. k Newly formed, short procentrioles in G2-arrested ALMS1∆ex8 cells were quantified for their locations (left) and orientations (right) relative to the host centriole. Duplicated centrioles in G2-arrested WT cells were used as the control (ctrl). The procentriole location was marked on the Y-axis using mother centrioles as the matrix, which is normalized to one unit. Procentrioles formed below position 0 (dashed line) were seen in ALMS1∆ex8 cells. The angle between the procentriole and mother centriole was measured using mean±2 s.d. from the control as the normal range (dashed lines). Data are mean ± s.d. and analyzed by two-tailed Welch’s t-test (****P < 0.0001); samples were collected evenly from 3 biological replicates. Source Data file is provided. l Summary of ALMS1∆ex8-induced positive feedback loops in which the cartwheel and centriole impose each other’s amplification (see the discussion). In brief, upon G2 arrest, the expanded cartwheel inside the daughter splits into two units, with one shedding from the lumen. The shed unit, tethered to the nearby mother centriole, grows into a granddaughter centriole inside which the cartwheel expands again, while the daughter centriole carrying the other cartwheel unit disengages and drifts away, leading to the 3-x-x or 4-x-x configurations. This “expand-split-shed-grow” process occurs again at the newly seeded granddaughter, leading to 5-x-x or 6-x-x configurations. Solid arrows indicate cartwheel splitting and shedding accompanying centriole disengagement, and dashed arrows indicate procentriole and cartwheel growth.

Fig 3: ALMS1 mediates the cyclical assembly-disassembly dynamics of CEP152-CEP63-PCNT composites that function as the seed for cartwheel formation.a Immunoprecipitation (IP) of the endogenous ALMS1 or CEP63 from either control cells (NF2-/-; p53-/-) or cartwheel-KO cells (STIL-/-). Antibodies used for IP and western blot were indicated. Note that before IP, the endogenous CEP63 in the whole cell extract (WCE) was undetectable, as reported previously. Western blot source data are provided as a Source Data file. b UExM images of mature centrioles showing non-colocalizations of ALMS1 with CEP152, CEP63, or PCNT. c CEP152 puncta formed in S or G2 and unformed during mitosis. Quantification of CEP152 puncta in indicated cartwheel-KO cell lines. Cdt1 indicates G1, BrdU marks S, and G2 cells lack both. Data are mean ± s.d., analyzed by two-tailed Welch’s t-test (****P < 0.0001). Source data are provided as a Source Data file. d CEP152 formed composite structures with CEP63 but not with ALMS1. Images shown here were from STIL-/- cells. Other co-localization studies were shown in the Supplementary Fig. S3b. e CEP152 composites in STIL-/- or SAS-6-/- cells can robustly recruit PLK4. f CEP152 composites drive cartwheel or SAS-6 assembly. PLK4as cells inducibly expressing analog-sensitive PLK4 in the presence of 3MBPP1 form CEP152 composites (top panel). Upon washout of 3MBPP1 for 1 h, the cartwheel signal emerges from the CEP152 composite (bottom panel). g UExM images of SAS-6 assembly initiated from the CEP152 (top) or CEP63 puncta (bottom) 1 h after 3MBPP1 washout as described in (f). h ALMS1 is timely recruited to CEP152 composites upon mitotic entry, prior to their disassembly, in a PLK1-dependent manner. Asynchronously growing STIL-/- cells treated with or without PLK1 inhibition (200 nM BI-2536) for 6 hours were fixed. Cells just entering mitosis in their early prophase were examined for CEP152 and ALMS1 localization. Additional images revealing timely recruitment of ALMS1 during G2/M were shown in Supplementary Fig. S3c. i PLA reactions detecting the interaction of ALMS1 with CEP152 or CEP63 in interphase and mitotic cells. A representative image of PLA reactions that detect interactions between ALMS1 and CEP152 (ALMS1 x CEP152) in a pair of mitotic and interphase STIL-/- cells that co-stained with CEP152 composites. CEP152-/- cells were used as the negative control for PLA reactions. PLA signals for ALMS1 interaction with CEP152 or CEP63 in interphase and mitosis were quantified (right). See also the Supplementary Fig. S3d. Data are mean ± s.d., analyzed by two-tailed Welch’s t-test (****P < 0.0001). Source data are provided as a Source Data file. j The disassembly of CEP152 composites or CSs in mitosis requires PLK1 activities. Interphase SAS-6-/- cells obtained by shaking off mitotic cells were allowed to enter and arrest in mitosis in the presence of nocodazole alone ( + Noc) or nocodazole and BI-2536 ( + Noc +BI) for 6 h. Mitosis-arrested cells were then examined. Data are mean ± s.d., analyzed by two-tailed Welch’s t-test (****P < 0.0001). Source data are provided as a Source Data file. k Depletion of ALMS1 abolished CS assembly without impacting the level of CS components CEP152, CEP63, and PCNT. SAS-6-/- cells stably introduced with IPTG-inducible shRNA targeting ALMS1 were generated and used. Six days after IPTG induction, CS assembly was examined and quantified in S or G2 cells, as marked by cyclin B1 expression. Data are mean ± s.d., analyzed by two-tailed Welch’s t-test (****P < 0.0001). CS components before and after ALMS1 knockdown were analyzed by western blot (right). CEP63 was detected after IP. Source data, including Western blot films, is provided as a Source Data file. All DNA was shown in blue (DAPI). All scale bars are 1 μm (white) and 5 μm (yellow).

Fig 4: ALMS1 depletion and addback initializes centriole biogenesis anew, creating diverse yet heritable architectures that evolve through selection.a Canonical centrioles formed in variable numbers after NF2-/-; p53-/- cells were treated with the PLK4 inhibitor Centrinone for 10 days, followed by drug washout for 5 days. b Centrioles with diverse shapes or sizes emerged in the same cell after ALMS1 depletion and addback (ALMS1 restoration). NF2-/-; p53-/- cells carrying two DOX-inducible shRNA constructs targeting ALMS1 were treated with DOX for 7 days and reseeded in the absence of DOX for 5 days. c Two pairs of duplicated centrioles with extreme differences in size co-existing in early prophase. Note the size difference reflects the PCM size (γ-tubulin; red). d Applying selection pressure for tiny centrioles through a series of clonal cell isolations. Left, a diagram summarizing 5 runs of clonal cell isolation. In each run, the clone with the highest frequency of forming tiny centrioles was selected (circle). Middle, quantification of the centriole size in each selected clone, inferred by the length and width (L*W). After 5 runs, clone 80-23-26-8-44 grows a heterogeneous group of tiny centrioles all smaller than WT centrioles. Data are mean ± s.d., analyzed by bidirectional Welch’s t-test (****P < 0.0001). Source data are provided as a Source Data file. Right, ALMS1 levels were restored in each isolated clone, with α-tubulin as the loading control. e Representative UExM images of tiny centrioles from clone 80-23-26-8-44 cells that form partial cylinder shapes, the diameter of which is distinct from that of control centrioles. f UExM images of 3 tiny centrioles with different shapes co-existing in the same cell from clone 80-23-26-8-44. g, h Transmission electron microscopy (TEM) analyses of control or tiny centrioles. g One control and six tiny centrioles (#1-#6) were shown, including serial sectioning of three tiny centrioles (#4-#6). #4 tiny centriole appears to have 3 microtubule blades in two consecutive sections. Red boxes marked the width of WT centrioles for comparison. h One control and one tiny centriole in cross section were shown. Note that the tiny centriole has three microtubule triplets arranged at a ~ 70-degree angle instead of the ~40-degree angle for control centrioles. Red boxes marked the width of WT centrioles. UExM images of WT and tiny centrioles from (e) were placed below the EM image for easy comparison. i Clone 80-23-26-8-44 cells going through PLK4 (top) or ALMS1 (bottom) restoration were examined for centriole size. Larger centrioles capable of recruiting appendages formed and co-existed with tiny centrioles 5-6 days after ALMS1 restoration but not PLK4 restoration. j Quantification of 80-23-26-8-44 cells hosting appendage-equipped centrioles before and after ALMS1 restoration (80-23-26-8-44-R). After 3 runs of clonal cell isolation, a clone (80-23-26-8-44-R-3-6-15) where >95% of cells carry appendage-equipped centrioles was isolated. Data are mean ± s.d., with N = 3 independent biological replicates. Source data is provided as a Source Data file. k A model summarizing (i) the cartwheel-centriole relationship seen under the ALMS1∆ex8 condition and (ii) the ALMS1-mediated CS assembly-disassembly cycle, each capable of driving reproducible centriole biogenesis (see the Discussion).