Biology subjects

Sirajuddin, M.

Publications and source records attributed to Sirajuddin, M..

8 recordsLinked to original sources

bioRxiv2026

Glutamylated vimentin proteoform identified by a synthetic binder links to epithelial-mesenchymal plasticity

Cytoskeletal protein expression and filament dynamics change significantly during cell state transitions. However, post-translational modifications of cytoskeletal proteins during these transitions have rarely been described. Here, using a synthetic glutamylation-binder (SB2B49) selected against a bi-glutamylated peptide epitope, we identify a distinct pool of glutamylated vimentin filaments. We demonstrate that vimentin glutamylation is enzymatically added by tubulin tyrosine ligase-like (TTLLs) and removed by cytosolic carboxypeptidases (CCPs). Mass spectrometry and mutagenesis reveal that glutamylation occurs on specific vimentin residues. We find that glutamylated vimentin levels are dynamically modulated during epithelial-mesenchymal plasticity. During collective migration in scratch-wound assays, glutamylated vimentin filaments are transiently depleted at the wound edge, a process controlled by canonical glutamylation writer-erasers. Our findings reveal a new layer of vimentin regulation via glutamylation and establish the glutamylation-binder as a valuable tool for exploring the diversity of vimentin proteoforms and glutamylation modifications in both physiological and pathological contexts.

cell biology↗

bioRxiv2026

Tubulin glycylation regulates microtubule-protein interactions that are key for ciliary stability and trafficking

Tubulin glycylation, a cilia-specific posttranslational modification is emerging as a potentially key regulator of ciliary axonemal microtubules. However, insights into the functional consequences of glycylation have remained limited. Here, using in vitro reconstitution assays with unmodified or custom-glycylated tubulin, we provide a systematic mechanistic analysis of glycylation-dependent regulation of motors and microtubule-associated proteins. Our studies highlight that glycylation selectively enhances ciliary kinesin-2 motility while reducing kinesin-1 activity, suggesting a role in promoting efficient intraflagellar transport along axonemal microtubules. Moreover, glycylation protects microtubules from decay by suppressing the activities of the depolymerase MCAK and severing enzyme spastin, thereby enhancing stability. Notably, this regulation is dependent on the proportion of glycylation on the microtubule surface, coupled with concomitant reduction of glutamylation. Thus, by generating microtubule surfaces with distinct biochemical states, we establish that combinatorial modification patterns define functional microtubule properties especially in cilia. Together, our findings provide the first comprehensive mechanistic framework for tubulin glycylation in regulating molecular motors and MAPs in cilia, establishing glycylation as a key determinant of motor selectivity and microtubule stability within the axoneme.

biochemistry↗

bioRxiv2022

Doublet microtubule inner junction protein FAP20 recruits tubulin to the microtubule lattice

Motility of organisms involves beating of cilia and flagella that are composed of doublet microtubules. The doublets are made of A- and B-tubules that fuse together at two junctions. Among these, the outer junction is made of tripartite tubulin connections, while the inner junction contains distinct non-tubulin elements. The latter includes Flagellar-associated protein 20 (FAP20) and Parkin co-regulated gene protein (PACRG) that together link the A- and B- tubules at the inner junction. While the structures of doublet microtubules reveal molecular details, their assembly is poorly understood and examining proteins at the junctions can provide important clues. In this study, we purified recombinant FAP20 and characterized its effects on microtubule dynamics by TIRF microscopy. Using in vitro reconstitution and cryo-electron microscopy, we conclusively show that FAP20 recruits free tubulin to the existing microtubule, where it mediates flexible lateral interactions between the microtubule lattice and tubulin dimers. Our structure of microtubule:FAP20:tubulin complex partially resembles the inner junction architecture, further providing insights into assembly steps involved in closure of B- tubule in a doublet microtubule.

biophysics↗

bioRxiv2022

Myofiber reconstruction at micron scale reveals longitudinal bands in heart ventricular walls

The coordinated contraction of myocytes drives the heart to beat and circulate blood. Due to the limited spatial resolution of whole heart imaging and the piecemeal nature of high-magnification studies, a confirmed model of myofiber geometry does not yet exist. Using microscopy and computer vision we report the first three-dimensional reconstruction of myofibers across entire mouse ventricular walls at the micron scale, representing a gain of three orders of magnitude in spatial resolution over the existing models. Our analysis reveals prominent longitudinal bands of fibers that are orthogonal to the well-known circumferential ones. Our discovery impacts present understanding of heart wall mechanics and electrical function, with fundamental implications for the study of diseases related to myofiber disorganization.

cell biology↗

bioRxiv2022

Peptide derived nanobody inhibits entry of SARS-CoV-2 variants

Emergence of the new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS variants and other viruses in a short period of time becomes essential. Blocking the SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-ACE2 interaction has been already exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a 9 amino acid peptide from the receptor-binding motif (RBM) of Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the nanobody. In summary, our study presents a faster and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. This approach can also be widely extended to target other viruses and pathogens in the future.

cell biology↗

bioRxiv2021

Initiation of ensemble kinesin-3 motility is regulated by the rigidity of cargo-motor attachment

Intracellular cargo transport is powered by molecular motors that move on their respective filamentous tracks. A key component in this process is the tether between cargo and motor, which is often connected by long slender coiled-coils. Several studies have identified mechanisms that regulate cargo transport and can be broadly categorized into regulation of the motor ATPase activity by autoinhibition, cargo adapters and modifications in the cytoskeletal tracks. The regulatory effects of cargo-motor linkers have been described in kinesin-3 subfamily motors. However, the effects of cargo-motor linker rigidity on ensemble cargo transport has not been explored. Here we have built a DNA origami scaffold, which can be tethered with multiple kinesin-3 motors using either single or double-stranded DNA linkages, mimicking rigid versus flexible cargo-motor linkages. Using this system, we show that regardless of the motor numbers attached to the cargo, only linkers with a lesser degree of freedom allow motors to engage with microtubule tracks. Together, our work identifies that the rigidity of cargo-motor linkages influences motor motility. This opens up the possibilities to identify new factors that can influence the rigidity of cargo-motor linkages that in turn can regulate intracellular cargo transport.

biochemistry↗

bioRxiv2020

Genetically encoded live cell sensor for tyrosinated microtubules

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin post-translational modification (PTMs). These PTMs are known to affect microtubule stability, dynamics and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present there exist no tool that can specifically mark tubulin PTMs in live cells, thus severely limiting our understanding of tubulin PTMs. Using yeast display library, we identified a binder against terminal tyrosine of alpha tubulin, a unique PTM site. Extensive characterization validates the robustness and non-perturbing nature of our binder as tyrosination sensor, a live cell tubulin nanobody specific towards tyrosinated or unmodified microtubules. Using which, in real time we followed nocodazole, colchicine and vincristine induced depolymerization events of unmodified microtubules, and found each distinctly perturb microtubule polymer. Together, our work describes the tyrosination sensor and potential applications to study microtubule and PTM processes in living cells.

cell biology↗

bioRxiv2019

Structural insights into filament recognition by cellular actin markers

Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

cell biology↗