Targeting cytoskeletal phosphorylation in cancer

Phosphorylation of cytoskeletal proteins regulates the dynamics of polymerization, stability, and disassembly of the different types of cytoskeletal polymers. These control the ability of cells to migrate and divide. Mutations and alterations of the expression levels of multiple protein kinases are hallmarks of most forms of cancer. Thus, altered phosphorylation of cytoskeletal proteins is observed in most cancer cells. These alterations potentially control the ability of cancer cells to divide, invade and form distal metastasis. This review highlights the emergent role of phosphorylation in the control of the function of the different cytoskeletal polymers in cancer cells. It also addresses the potential effect of targeted inhibitors in the normalization of cytoskeletal function.

Implicit Laplacian of Enhanced Edge (ILEE): An unguided quantitative cytoskeletal imaging algorithm

The eukaryotic cytoskeleton plays essential roles in cell signaling, trafficking, and motion. Recent work towards defining the temporal and spatial dynamics of cytoskeletal organization, including as a function of cell status, has utilized quantitative analysis of cytoskeletal fluorescence images as a standard approach to define cytoskeletal function. However, due to the uneven spatial distribution of the cytoskeleton, including varied shape and unstable binding efficiency to staining markers, these approaches may not segment cytoskeletal fractions accurately. Additionally, quantitative approaches currently suffer from human bias as well as information loss caused by z-axis projection of raw images. To overcome these obstacles, we developed Implicit Laplacian of Enhanced Edge (ILEE), a cytoskeletal component segmentation algorithm, which uses an 2D/3D-compatible, unguided local thresholding approach, therefore providing less biased and stable results. Empowered by ILEE, we constructed a Python based library for automated quantitative analysis of cytoskeleton images, which computes cytoskeletal indices that covers density, bundling, severing, branching, and directionality. Comparing to various classic approaches, ILEE library generates descriptive data with higher accuracy, robustness, and efficiency. In addition to the analysis described herein, we have developed an open-access ILEE library for community use.

A cytoskeletal function for PBRM1 reading methylated microtubules

Epigenetic effectors “read” marks “written” on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

The chromatin modifier SETD2 was recently shown to be a dual-function methyltransferase that “writes” methyl marks on both chromatin and the mitotic spindle, revealing α-tubulin methylation as a new posttranslational modification of microtubules. Here, we report the first cytoskeletal “reader” for this SETD2 methyl mark: the polybromo protein PBRM1. We found PBRM1 directly binds the α-Tub-K40me3 mark on tubulin, and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 can assemble a PBAF complex in the absence of chromatin as revealed by mass spectrometry, and can recruit other PBAF complex components including SMARCA4 and ARID2 to α-tubulin. In addition to PBRM1, other PBAF components were also localized to the mitotic spindle and spindle pole. This PBAF localization was dependent on recruitment to microtubules by PBRM1, and loss of spindle-associated PBRM1/PBAF led to genomic instability as assessed by increased formation of micronuclei. These data reveal a previously unknown function for PBRM1 beyond its role remodeling chromatin, and expand the repertoire of chromatin remodelers involved in writing and reading methyl marks on the cytoskeleton. The results of this study lay the foundation for a new paradigm for the epigenetic machinery as chromatocytoskeletal modifiers, with coordinated nuclear and cytoskeletal functions.