Biology subjects

Mali, G. R.

Publications and source records attributed to Mali, G. R..

5 recordsLinked to original sources

bioRxiv2026

Inhibition of CDK7, CDK9 or CDK12 affects phosphorylation of human SPT5.

SPT5, complexed with SPT4 in the DRB-sensitivity-inducing factor, DSIF, is a critical regulator of transcription elongation that acts negatively in the unphosphorylated form and positively when phosphorylated. In vivo, phosphorylation of multiple sites in the largely unstructured linker and C-terminal regions of the protein facilitates release from an early elongation checkpoint and processive transcription elongation. Transcription elongation factors, including the PAF complex (PAF1C), interact with phosphorylated SPT5 and phosphorylation of residues S666 and T806 are associated with the positive elongation functions of SPT5. Here, using in vitro phosphorylation of regions of the SPT5 C-terminus followed by GST-mediated pull-down we confirm that the RTF1 subunit of PAF1C interacts with residues 750-850 only when phosphorylated. Antibodies we raised to phospho-S666 or phospho-T806 are specific for these sites phosphorylated in vitro and in vivo. Western analysis of endogenous SPT5 in HEK293 cells using these antibodies before and after inhibition of analog-sensitive versions of CDK7, CDK9 and CDK12 indicate that all three kinases play roles in the phosphorylation of SPT5, although inhibition of CDK9 has the most marked effect. CDK9 inhibition also inhibits the association of PAF1C subunits with SPT5 in vivo and affects phosphorylation of chromatin-associated SPT5 as measured by chromatin immunoprecipitation (ChIP).

molecular biology↗

bioRxiv2026

Motile ciliophagy promotes ciliary recycling under stress

Motile cilia on eukaryotic cell surfaces are exposed to a wide array of external stresses. Ciliary remodeling in response to stress remains understudied despite being pivotal to cell homeostasis. Here, we apply a commonly used calcium perturbation to mimic hyper-osmotic cellular stress in the model ciliate Tetrahymena thermophila. We find that partially deciliated Tetrahymena, upon calcium shock, internalise entire ciliary axonemes into ring-like configurations we refer to as c-rings. Partially deciliated cells recover and regenerate new cilia over time. We propose that the recycling of c-rings releases axonemal building blocks to allow ciliary regeneration. We use time-course ultrastructure expansion microscopy (U-ExM) to show that the ciliary tubulin code undergoes a dynamic pattern of erasure during the bulk disassembly of doublet microtubules within the c-rings. Using whole cell quantitative proteomics, we find that partial deciliation induces the autophagy of c-rings and activates de novo dynein synthesis by upregulating several axonemal dynein assembly factors (DNAAFs). Transmission electron microscopy (TEM) and confocal imaging confirms that intact ciliary axonemes are encapsulated within VPS13A +ve autophagic vacuoles. We propose that internalised motile cilia undergo a regulated process of bulk degradation we term "motile ciliophagy" for the recycling of axonemal components for cilia regeneration. Our work establishes a link between ciliary turnover via macroautophagy and cellular homeostasis which may be a conserved response to stress.

cell biology↗

bioRxiv2024

DNAAF9 (Shulin) and ARL3 regulate the transport and activation of ciliary Outer Dynein Arms

Multiciliogenesis requires large-scale biosynthesis of motility-powering axonemal inner and outer dynein arm motors (IDA and ODA) prior to their intraflagellar transport (IFT) into cilia. ODAs are inhibited by the packaging chaperone Shulin during ciliogenesis in T. thermophila. How Shulin is released for ODAs to become active inside cilia remains unclear. We establish interactions between DNAAF9 (human Shulin) and mammalian ODA subunits, IFT proteins and the ciliary small GTPase ARL3 using proteomics and in vitro reconstitutions. Mutagenesis combined with biochemical and structural studies reveal that DNAAF9 and Shulin preferentially bind the active Arl3-GTP state. GTP-loaded Arl3 can access, bind and displace Shulin from the packaged ODA-Shulin complex. We propose that once the inhibited ODA complex enters growing cilia, Arl3-GTP displaces Shulin (DNAAF9) and sequesters it away from ODAs promoting activation of their motility specifically inside cilia.

cell biology↗

bioRxiv2020

Shulin packages axonemal outer dynein arms for ciliary targeting

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During cilio-genesis, these [~]1.8 MDa complexes are assembled in the cytoplasm and targeted to cilia via an unknown mechanism. Here we use the ciliate Tetrahymena to identify two novel factors (Q22YU3 and Q22MS1) which bind ODAs in the cytoplasm and are required for their delivery to cilia. We show that Q22YU3, which we name Shulin, locks the ODA motor domains into a closed conformation and inhibits motor activity. Cryo-EM reveals how Shulin stabilizes this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

molecular biology↗

bioRxiv2017

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of substrate client proteins. How the ubiquitous chaperone machinery directs its activities towards a specific set of substrates and whether this selectivity could be targeted for therapeutic intervention is of intense research. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone for the FKBP8-HSP90 chaperone complex during the biosynthesis of axonemal dynein heavy chains required for cilia motility. In the absence of ZMYND10, defects in dynein heavy chains trigger broader dynein motor degradation. We show that FKBP8 inhibition phenocopies dynein motor instability in airway cells, and human disease-causing variants of ZMYND10 disrupt its ability to act as FKBP8-HSP90 co-chaperone. Our study indicates that the motile ciliopathy Primary Ciliary Dyskinesia (PCD) should be considered a cell-type specific protein-misfolding disease and opens the potential for rational drug design that could restore specificity to the ubiquitous chaperone apparatus towards dynein subunits.

cell biology↗