Biology subjects

Balasubramanian, M. K.

Publications and source records attributed to Balasubramanian, M. K..

6 recordsLinked to original sources

bioRxiv2025

Fluorescent protein tags for human tropomyosin isoform comparison

Tropomyosin is an important actin cytoskeletal protein underpinning processes such as muscle contraction, cell shape and cell division. Defects in tropomyosin function can lead to diseases, including some myopathies and allergies. In cells, tropomyosin molecules form coiled-coil dimers, which then polymerise end-to-end with other dimers for actin association. Tropomyosin is challenging to tag for in vivo fluorescence microscopy without perturbing its polymerisation interfaces. We recently developed a fluorescent tag comprising a forty amino acid flexible linker capable of detecting tropomyosin in S. pombe actin cables and the actomyosin ring, and in patch-like structures that were previously unappreciated. We also used this strategy successfully to tag human TPM2.2, a prominent human muscle isoform. Here, we expand this tool to visualise eight other human tropomyosin isoforms, using mNeonGreen, mCherry and mStayGold tags. All show typical tropomyosin fluorescence, no signs of cytotoxicity and are compatible with super-resolution microscopy. These tools singly or in combination should aid detailed mechanistic investigations of tropomyosin isoforms. Summary StatementGreen and red fluorescent fusion proteins with lengthy and flexible polypeptide linkers readily allow improved visualisation and comparison of tropomyosin isoform distribution in live human cells.

cell biology↗

bioRxiv2024

StayRose: a photostable StayGold derivative red-shifted by genetic code expansion

Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric green fluorescent protein recently monomerised through sequence engineering, addresses this challenge with its high photostability. There is now focus on producing different colour StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to red-shift super folder GFP upon incorporation into its chromophore via genetic code expansion. Here we apply the same strategy to red-shift StayGold through substitution of Tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows 530 nm excitation and 588 nm emission peaks, shifting from the 497 nm and 504 nm excitation and emission peaks of StayGold. StayRose also retains the favourable photostability of StayGold and can be similarly monomerised using mutations at the dimer interface. A high-resolution crystal structure of StayRose confirms the modified structure of the amino-chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step towards developing red-shifted StayGold derivatives.

bioengineering↗

bioRxiv2022

Structural Mechanisms of Actin Isoforms

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle changes in their design principles determine the interaction with myosin motors and actin-binding proteins. The functional diversity is further increased by posttranslational modifications (PTMs). Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal -actin (3.37[A]), cardiac -actin (3.07[A]), {beta}-actin (2.99[A]), and {gamma}-actin (3.38[A]) in the Mg2+{middle dot}ADP state with their native PTMs. The structures revealed isoform-specific conformations of the N-terminus that shifts closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Retropropagated structural changes further show that myosin binding modulates actin filament structure. Further, our structures enabled us to reveal the location of disease-causing mutations and to analyze them with respect to known locations of PTMs. Collectively, the previously unknown structures of single-isotype, posttranslationally modified bare cardiac -actin, {beta}-actin, and {gamma}-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

biochemistry↗

bioRxiv2019

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Diazirine-lysine, Bicyclononyne-lysine and Azido-lysine

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

synthetic biology↗

bioRxiv2019

Polar relaxation by dynein-mediated removal of cortical myosin II

Nearly 6 decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction (White and Borisy, 1983; Wolpert, 1960). While this mechanism has remained controversial (Rappaport, 1996), recent work has provided evidence for polar relaxation by astral microtubules (Chen et al., 2008; Dechant and Glotzer, 2003; Foe and Dassow, 2008; Murthy and Wadsworth, 2008; Werner et al., 2007), although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction whereas suppression of dynein activity delayed furrowing. We provide evidence that dynein-mediated removal of myosin II from the polar cortexes triggers cortical flow towards the cell equator, which induces the assembly of the actomyosin contractile ring. These studies for the first time provide a molecular basis for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

cell biology↗

bioRxiv2018

Stabilising and destabilising kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends

In fission yeast, the length of interphase microtubule (iMT) arrays are adapted to cell length so as to maintain cell polarity and to help centre the nucleus and cell division ring. Here we show that length regulation of iMTs is dictated by spatially-regulated competition between MT-stabilising Tea2/Tip1/Mal3 (Kinesin-7) and MT-destabilising Klp5/Klp6/Mcp1 (Kinesin-8) complexes at iMT plus tips. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus tips of iMT bundles and restricts access to the plus tips by Klp5/Klp6/Mcp1, which accumulates behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus tips and MT catastrophe. These data show that in vivo, whilst the \"antenna model\" for iMT length- and age-dependent catastrophase accumulation has validity, length control is an emergent property reflecting spatially-regulated competition between multiple complexes at the MT plus tip.

cell biology↗