Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

Protocol to measure centrosome cohesion deficits mediated by pathogenic LRRK2 in cultured cells using confocal microscopy

“… Note: The same protocol as outlined here is applicable to HEK293T, SH-SY5Y, primary murine astrocytes, primary human dermal fibroblasts, lymphoblastoid cell lines, iPSCs and iPSC-derived neural precursor cells (NPCs). 1 , 7 , 8 , 9 Optimal fixation and permeabilization conditions for other cell types need to be independently determined. …”

“…For complete details on the use and execution of this protocol, please refer to Fdez et al. 1 and Lara Ordóñez et al. 2 …”

The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization

“…Such dynamic behavior is consistent with the direct RILPL1-mediated recruitment of phospho-Rab8/10 to a centriolar location to cause an increase in the distance between duplicated centrosomes. It further predicts that the underlying mechanism may involve the dynamic displacement of protein(s) necessary to keep the duplicated centrosomes in close proximity to each other ( Fdez et al, 2022 ), perhaps via steric hindrance.…”

“…Phospho-Rab10 staining largely overlaps with tagged RILPL1 in A549 cells over-expressing pathogenic LRRK2 and partially overlaps with a centrosomal marker in MEFs endogenously expressing pathogenic LRRK2. Since it is the membrane-bound active form of Rab10 which is phosphorylated by LRRK2 ( Liu et al, 2018 ; Gomez et al, 2019 ; Lara Ordóñez et al, 2019 ; Fdez et al, 2022 ), it will be interesting to determine whether membrane-bound vesicular structures containing phospho-Rab10 accumulate at the subdistal appendage of the mother centriole in pathogenic LRRK2-expressing cells. In addition, and given recent reports of the localization of active Rab10 at the mother centriole in a manner regulated by the GDP/GTP exchange factor DENND2B ( Kumar et al, 2022 ), future studies should address whether the pathogenic LRRK2-mediated centriolar phospho-Rab10 accumulation and the resulting cohesion deficits are modulated by DENND2B.…”

“…In U2OS cells, around 90-95% of cells display a distance between duplicated centrosomes <2 µm, and centrosome splitting in this cell type has been defined when the duplicated centrosomes are >2 µm apart ( Meraldi and Nigg, 2001 ). Performing identical analyses by measuring the distances between duplicated centrosomes from around 100 non-transfected wildtype cells, centrosomes were scored as being split when the distance between their centers was >1.5 µm for HEK293T cells, >2.5 µm for A549 cells, or >5 µm for MEF cells, respectively ( Madero-Pérez et al, 2018 ; Lara Ordóñez et al, 2019 ; Fdez et al, 2022 ). For each condition and experiment, distances were scored from around 30 transfected cells with duplicated centrosomes (HEK293T and A549 cells) or from around 50 cells (MEFs) with duplicated centrosomes, and mitotic cells were excluded from the analysis in all cases.…”