Hypothesis

In aged, slowly cycling somatic cells, centrosome clustering does more than generate merotelic kinetochore‑microtubule attachments; it creates a concentrated pericentriolar‑material platform that chronically activates the Hippo pathway, leading to sustained YAP phosphorylation, cytoplasmic retention, and a senescence‑associated secretory phenotype (SASP). This signaling output, rather than chromosome missegregation, is the primary driver of tissue‑level dysfunction in aging.

Rationale

• Centrosome clustering is a tension‑dependent process that relies on KIFC1, astral microtubule‑cortex interactions, and spindle‑associated proteins (e.g., TACC3‑ILK, augmin) KIFC1-dependent clustering. When supernumerary centrosomes are forced into two poles, the pericentriolar material (PCM) from each centrosome is brought into close apposition, increasing the local concentration of PCM‑resident signaling molecules.
• Centrosomes function as signaling hubs that can modulate the Hippo pathway via RASSF1A‑MST1, regulate proteasome substrates such as p53, and trigger PIDDosome activation Centrosome as Hippo signaling hub. Clustering multiple centrosomes therefore amplifies the density of these Hippo‑regulatory scaffolds.
• Aged somatic cells exhibit persistent centrosome amplification without frequent multipolar divisions, correlating with tissue‑specific chromosomal instability Centrosome amplification in aged cells. Yet direct evidence linking clustering to functional outcomes beyond CIN is lacking.
• Hippo/YAP signaling is a known regulator of cellular senescence and SASP; sustained Hippo activation leads to YAP cytoplasmic sequestration, promoting a pro‑inflammatory secretome that contributes to aging phenotypes.

Thus, we propose that the dominant consequence of centrosome clustering in aged, non‑proliferating cells is aberrant Hippo signaling, which drives senescence and tissue decline, while CIN remains a secondary, less impactful byproduct.

Testable Predictions

1. In aged human fibroblasts or mesenchymal stem cells, pharmacological or genetic inhibition of centrosome clustering (e.g., KIFC1 siRNA or monastrol‑resistant KIFC1 mutant) will reduce merotelic attachments (measured by kinetochore‑microtubule tension assays) but increase Hippo pathway activity (↑ phospho‑LATS1/2, ↑ phospho‑YAP127, ↓ nuclear YAP).
2. Conversely, forcing centrosome declustering (via overexpression of a dominant‑negative KIFC1 or depletion of augmin subunit HAUS6) will diminish Hippo signaling readouts and lower SASP factor secretion (IL‑6, IL‑8, MMP‑3) despite a rise in lagging chromosomes.
3. Rescue experiments: Re‑expressing a constitutively active YAP (YAP‑5SA) in clustered‑centrosome cells will suppress SASP expression even when Hippo signaling is high, indicating that YAP inactivation is necessary for the senescence phenotype.
4. In vivo validation: Aged mouse liver or muscle tissue treated with a centrosome‑declustering peptide will show reduced p‑YAP immunostaining and lower SASP markers in situ, without a significant change in aneuploidy rates measured by single‑cell sequencing.

Experimental Design

• Cell model: Passaged human dermal fibroblasts (>20 population doublings) exhibiting centrosome amplification (γ‑tubulin foci >2 per nucleus).
• Interventions: (a) KIFC1 siRNA; (b) CRISPRi of HAUS6; (c) control non‑targeting siRNA.
• Readouts:
  • CIN: Live‑cell imaging of H2B‑mCherry to score lagging chromosomes; fixed‑cell CREST staining for merotelic attachments (kinetochore bound by α‑tubulin from both poles).
  • Hippo/YAP: Western blot for p‑LATS1/2, p‑YAP127, total YAP; immunofluorescence for nuclear vs cytoplasmic YAP; luciferase reporter (8xGTIIC‑Luc) for transcriptional activity.
  • SASP: ELISA/qPCR for IL‑6, IL‑8, MMP‑1, MMP‑3.
  • Centrosome status: γ‑tubulin and pericentrin staining to quantify clustering index (distance between centrosome pairs).
• Controls: Young fibroblasts (passage 5) and cancer cell line with known CIN (e.g., HCT116) to verify assay specificity.

Potential Outcomes and Interpretation

• If Hippo signaling rises upon declustering while CIN increases, the hypothesis is supported: clustering suppresses a deleterious Hippo response, and its loss unmasks a signaling‑driven senescence phenotype.
• If Hippo activity remains unchanged or decreases upon declustering, the hypothesis would be refuted, suggesting that centrosome clustering’s primary impact is indeed CIN‑centric or that Hippo regulation occurs via a different mechanism.
• If SASP correlates inversely with nuclear YAP levels irrespective of CIN magnitude, it reinforces the notion that Hippo/YAP dysregulation, not chromosome missegregation, drives the aging phenotype.

This framework directly links the biophysical act of centrosome clustering to a biochemical signaling cascade, offering a falsifiable, mechanism‑centric explanation for how aged somatic tissues deteriorate despite avoiding catastrophic multipolar divisions.