Research Program
Polyglutamylation and polyglycylation are key posttranslational modifications (PTMs) of tubulin highly enriched in axonemes, the core microtubule-based structure of mammalian cilia and flagella. While there is a semblance of understanding about their role in motile cilia, very little is known about what these PTMs do in primary cilia, key signalling centres of epithelial cells. There is very little understanding of the balance or interplay between glutamylation and glycylation and how altering either of them can lead to ciliopathies in humans. Both these modifications are suggested to have key roles in microtubule-protein interactions, a central aspect of ciliary trafficking, and thus, our focus is on understanding how a balance between either of these modifications regulates trafficking and thus, signalling processes in primary cilia and how this impacts proper cellular functions and tissue homeostasis using retina and skin as our current model organs. In this regard, we are specifically focussing on:​​​​​​
Decipher the role of tubulin glycylation in mammalian cilia
microtubules gliding on kinesin-1 K560
One of the long-standing question in the tubulin PTM field is what does glycylation do in cilia. While there is some understanding of its role in motile cilia and sperm flagella, this is still in its nascent stages. In terms of its role in primary cilia, there is little known. Here, we aim to understand how glycylation regulates specific aspects of cilia functions, like intraflagellar transport, length maintenance and regulation of specific ciliary signalling pathways through its modulation of the axonemal cytoskeleton.
To address this, we use in vitro microtubule-protein interactions as well as in cellulo approaches combining biochemistry, cell biology and advanced imaging. In this regard, we have established a toolbox of custom-modified tubulin for the in vitro analyses and a host of cell lines either overexpressing the glycylases under a ciliary localisation signal, or CRISPR/Cas9 KO for the enzyme for a holistic analysis of the altered balance of this modification on regulating cilia functions
Immunostaining of Rpe-1 cells for cilia and changes in intraflagellar transport upon modulating tubulin glycylation
Establish a mechanistic link between human retinal ciliopathies and perturbed tubulin PTMs
Immunostaining of Rpe-1 cells for cilia and tubulin glutamylation
The growing knowledge of how tubulin PTMs regulate microtubule functions emphasizes the need to identify specific human disorders caused by tubulin PTM defects, to develop proper therapeutics. In this regard, studies show that humans with mutations in specific glutamylases and deglutamylases have vision disorders. However, the underlying mechanisms are unknown.
In the laboratory, we aim to address this knowledge gap using a multi-modal approach of assessing the impact of the reported enzyme mutations on their activity, followed by their effect on primary cilia structure and function. While the mechanistic insights at the cellular level will be established using retinal pigment epithelial cell lines that will bear these mutations introduced using CRISPR-Cas9 gene editing, the long-term physiological role will also be investigated using stem cell-derived retinal epithelium and retinal organoids.
Study impact of tubulin PTMs on homeostasis
Tissue repair involves haemostasis, inflammation, proliferation and extracellular matrix remodelling, which ultimately leads to wound closure. Many skin cells involved here like the keratinocytes, endothelial cells, Langerhans cells and dermal fibroblasts are ciliated. Among them, fibroblasts are central players, and their primary cilia are involved in cytoskeletal reorganisation, cell polarity, directionality and speed of migration, acheived through multiple ciliary signalling pathways. These in turn depend on intraflagellar transport, suggesting a role for PTMs in tissue homeostasis. Moreover, when it comes to glutamylation and glycylation, there seems to be an intricate crosstalk and interplay between these PTMs, where altering one affects the other. This suggests that there is a very fine balance between these modifications and even a slight alteration can affect the overall cellular function and thus, organ and organism homeostasis.
Our goal is to first establish the PTM distribution in the skin and then assess the role of glutamylation and glycylation on homeostasis using diverse transgenic mouse models. We study aspects of cilia organisation, as well as their role in wound closure processes involving cell migration and signalling cascades. In the long term, our aim is to understand the overall crosstalk between cilia from different cells involved in homeostasis using co-culture or 3D assembloid models. These studies will provide cellular insights into how the two PTMs influence homeostasis, enabling further in vivo validation.
Identifying the spectrum of non-tubulin substrates of these TTLLs that can potentially regulate cell- and/or cilia functions
Tubulin is not the sole substrate for the PTM enzymes. Several ciliary and non-ciliary cellular proteins with distinct cellular functions can be modified by these enzymes. The identity of these proteins is yet not fully established, especially in the context of the cilia and flagella. While a few of the substrates for a couple of the glutamylases are identified, the substrates of glycylases are yet unknown. Moreover, the identity of the proteins modified within the cilia and flagella is yet to be established.
Combining molecular biology, biochemistry, cell biology and quantitative proteomic approaches, we are establishing a large dataset of proteins which can be potentially modified by the different tubulin PTM enzymes both in different ciliating cell lines as well as in transgenic knockout mouse models. We also aim to establish whether modifications of non-tubulin proteins influence the overall cellular and ciliary functions, and thus establish whether the observed ciliopathies due to PTM enzyme mutations are exclusive to imbalance of tubulin PTMs only, or of other proteins as well. This will open up the possibility that these enzymes, their modification patterns and their substrates can be potential targets for future therapeutics