Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The dynein motor protein complex is crucial for various cellular processes, including mitosis.
• Regulation of dynein's interaction with its diverse cargo is essential for its functions.
• The C-terminal domain (CTD) of the light intermediate chain 1 (LIC1) subunit is a key regulatory site for dynein cargo binding.

Purpose of the Study:

• To investigate the role of mitotic phosphorylation of LIC1-CTD in regulating dynein function during mitosis.
• To determine how LIC1-CTD phosphorylation affects dynein's interaction with specific protein complexes and its localization.
• To explore the functional consequences of LIC1-CTD phosphorylation and dephosphorylation in human cells and zebrafish.

Main Methods:

• Phosphorylation site analysis of LIC1-CTD using mass spectrometry.
• Site-directed mutagenesis to create phosphomimetic and non-phosphorylatable LIC1-CTD mutants.
• Immunofluorescence microscopy to assess dynein localization, kinetochore-microtubule attachments, and centrosome positioning.
• Biochemical assays to study the interaction of dynein complexes with Pin1 and other binding partners.
• Analysis of mitotic progression and chromosome congression in treated cells.
• Phenotypic analysis of zebrafish embryos with mutated LIC1-CTD.

Main Results:

• Mitotic phosphorylation of LIC1-CTD at three cdk1 sites is essential for proper mitosis, dynein kinetochore loading, and spindle assembly checkpoint inactivation in human cells.
• Phosphorylation recruits the prolyl isomerase Pin1 to specific dynein-Nde1-Lis1 complexes, but not to dynein-dynactin complexes.
• Dephosphorylation of LIC1-CTD disrupts dynein-Pin1 binding, leading to centrosome detachment, impaired chromosome congression, and Golgi fragmentation during mitosis.
• Phosphomutation of a conserved LIC1-CTD site in zebrafish causes developmental abnormalities.
• Interphase membrane transport remains unaffected by LIC1-CTD dephosphorylation.

Conclusions:

• Mitotic phosphorylation of LIC1-CTD acts as a critical switch to differentially regulate distinct dynein populations during cell division.
• This phosphorylation event is vital for accurate chromosome segregation and overall mitotic progression.
• The findings highlight the evolutionary conservation of LIC1-CTD phosphoregulation, underscoring its fundamental importance in cellular processes.