We investigate how cells assemble large multi-subunit motor proteins called axonemal dyneins which power the rhythmic beating motion of eukaryotic cilia and flagella. Our major focus is a newly discovered family of structurally and functionally diverse proteins called Dynein Axonemal Assembly Factors/DNAAFs which shepherd the folding and assembly of individual dynein subunits into functional macromolecular motors. To uncover the molecular mechanisms of DNAAFs, we combine biochemistry, integrative structural modeling, cell biology, and proteomic approaches.
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A) Respiratory cilia contain dynein motors that power ciliary beating motion. B) Cryo-EM guided structural model of a pre-assembled dynein motor bound by the packaging inhibitory factor Shulin/DNAAF9.
Model of the axonemal dynein assembly pathway regulated by 19 DNAAF proteins. Functional loss of DNAAFs leads to Primary Ciliary Dyskinesia (PCD), a severe incurable respiratory disease in humans.
Motile cilia are actively beating microtubular protrusions found on many cell surfaces. They enable critical biological functions ranging from sperm propulsion to clearing mucus in lungs. Loss of cilia motion results in a severe incurable disease called Primary Ciliary Dyskinesia (PCD) marked by respiratory complications at birth. Progressive decline in lung function due to inefficient mucus clearance and recurrent infections can be life-limiting. Patients also display other clinical complications including sub-fertility.
Axonemal dyneins, which come in two flavours – the inner and outer dynein arms (IDAs and ODAs), power the rhythmic beating motion of cilia. Dyneins are massive molecular motors, ex. a single ODA motor, comprised of ~20 distinct subunits, is ~2 MDa in size. How cells synthesise such large and biochemically complex machines is a fundamental cell biology question that our lab aims to address.
A novel protein family called dynein axonemal assembly factors/DNAAFs shepherd dynein biosynthesis from their folding to final transport into cilia. DNAAFs are structurally distinct and functionally non-redundant i.e., loss of any one of the nineteen DNAAFs found in humans can derail the entire biosynthetic pathway leading to PCD. Several DNAAFs bind one another and cooperate with chaperone proteins to facilitate dynein biosynthesis but the molecular details of how DNAAFs function are still unclear.
PCD remains incurable. Uncovering the underlying pathology to find new treatments requires mechanistic studies on DNAAFs. This is the lab’s major goal – to dissect the molecular and structural basis of axonemal dynein assembly by DNAAFs. For this, we integrate cell biology (fluorescence microscopy, endogenous cellular proteomics), biochemistry (complex isolation, reconstitutions) and structural biology (negative stain and cryo-electron microscopy, AlphaFold2-Multimer structure predictions, crosslinking mass spectrometry).
2023
Spokes and barrels tune the asymmetric beating of mammalian sperm flagella.
Mali, G.R.
Nature Structal & Molecular Biology – 30(3): 236-238.
2021
Shulin packages axonemal outer dynein arms for ciliary targeting.
Mali, G.R.*, Abid Ali, F.,* Lau, C.K.,* Begum, F., Boulanger, J., Howe, J., Chen, Z.A., Rappsilber, J., Skehel, M. and Carter, A.P.
Science – 371(6532): 910-916.
2018
ZMYND10 functions in a chaperone relay during axonemal dynein assembly
Mali, G.R.*, Yeyati, P.L.*, Mizuno, S., Dodd, D.O., Tennant, P.A., Keighren, M.A., Zur Lage, P., Shoemark, A., Garcia-Munoz, A., Shimada, A., Takeda, H., Edlich, F., Takahashi, S., von Kreigsheim, A., Jarman, A.P. and Mill, P.
eLife – 19;7: e34389.
2017
KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.
Yeyati, P.L., Schiller, R., Mali, G., Kasioulis, I., Kawamura, A., Adams, I.R., Playfoot, C., Gilbert, N., van Heyningen, V., Wills, J., von Kriegsheim, A., Finch, A., Sakai, J., Schofield, C.J., Jackson, I.J. and Mill, P.
J Cell Biol. – 216(4): 999-1013. doi: 10.1083/jcb.201607032.
2014
HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.
Diggle, C.P.,* Moore, D.J.,*, Mali, G.,* Sheridan, E., Jarman, A.P. and Mill, P.
PLoS Genet. – 10(9): e1004577. DOI: 10.1371/journal.pgen.1004577