Biology subjects

Aher, A.

Publications and source records attributed to Aher, A..

9 recordsLinked to original sources

bioRxiv2026

Frequency modulated semi-continuous wave Doppler ultrasound: A novel method for the detection of scatterer velocity and range using wearable ultrasound instrumentation

Doppler ultrasound imaging has become a key tool for clinicians and researchers to measure the velocity of blood and tissues within the human body. Presently pulsed wave and continuous wave Doppler imaging dominate the space as the two most prominent methods and are considered distinct from one another. However, these two techniques can also be considered as ends of a continuum along which Doppler imaging and phase tracking techniques exist. At the midpoint between these methods exists another underexplored Doppler imaging technique we refer to as frequency modulated semi-continuous wave (FMSCW) Doppler. We present a full technical overview of this technique, simulations exploring the impact of selected imaging parameters as well as how noise impacts imaging, and present three exemplary imaging sessions collected from human participants using FMSCW Doppler collected from a wearable ultrasound system. The results of the simulations evaluating the impact of imaging parameters demonstrate the trade-off between resolution in the range and velocity axis as well as the impact of scatterer range and speed on the measurement error. The three sets of images collected from human participants serve to demonstrate FMSCW Doppler used in a variety of wearable ultrasound applications including cardiovascular monitoring, rehabilitation assessment, and prosthetic device control. Furthermore, we discuss the relationship between FMSCW Doppler and isomorphic techniques from radar as well as potential inspiration for improvements for FMSCW Doppler inspired by the echolocation of bats.

bioengineering↗

bioRxiv2026

Structure of the mammalian ciliary transition zone microtubule doublet

The ciliary transition zone gates bidirectional protein trafficking to maintain the specialized ciliary proteome using microtubule doublets as a scaffold. While ciliary axonemal doublets are well-characterized, the molecular architecture of the transition zone doublet remains elusive. Here, we report the structure of the mammalian transition zone doublet from bovine tracheal cilia using cryo-electron tomography at 4.7-5.0 [A] resolution. The transition zone doublet is a structurally independent segment defined by an 8 nm-periodic arrangement of unique microtubule-inner proteins (MIPs) and microtubule-associated proteins (MAPs). We identify the calcium-binding protein CAPSL as a lumenal MIP that forms a pseudo-helical spiral and stabilizes microtubules in vitro. Furthermore, a dense MAP network on the A-microtubule surface clashes with intraflagellar transport (IFT) motor binding sites, suggesting anterograde IFT is directed to the B-microtubule for potential regulation by MAP9. Our work provides a structural framework for understanding gated ciliary transport and transition zone-linked human ciliopathies.

Cell Biology↗

bioRxiv2024

Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex

The {gamma}-tubulin ring complex ({gamma}-TuRC) is an essential multiprotein assembly, in which {gamma}-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The {gamma}-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the {gamma}-TuRC. However, the structural basis of the NEDD1-{gamma}-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human {gamma}-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the {gamma}-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric -helical assembly that contacts the lumen of the {gamma}-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down{gamma} -tubulin from cultured cells. The structure of the {gamma}-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the {gamma}-TuRC, but suggest that anchoring of {gamma}-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the {gamma}-TuRC during microtubule nucleation.

biochemistry↗

bioRxiv2023

Structure of the γ-tubulin ring complex-capped microtubule

Microtubules are composed of /{beta}-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale /{beta}-tubulin tracks for intracellular transport that align with, rather than spiral along, the filaments long-axis. We report that the human [~]2.3MDa {gamma}-tubulin ring complex ({gamma}-TuRC), an essential regulator of microtubule formation that contains 14 {gamma}-tubulins, selectively nucleates 13-protofilament microtubules. Cryo-EM reconstructions of {gamma}-TuRC-capped microtubule minus-ends reveal the extensive intra- and inter-domain motions of {gamma}-TuRC subunits that accommodate its actin-containing luminal bridge and establish lateral and longitudinal interactions between {gamma}- and -tubulins. Our structures reveal how free {gamma}-TuRC, an inefficient nucleation template due to its splayed conformation, transforms into a stable cap that blocks addition or loss of /{beta}-tubulins from minus-ends and sets the lattice architecture of cellular microtubules. One Sentence SummaryStructural insights into how the {gamma}-tubulin ring complex nucleates and caps a 13-protofilament microtubule.

biochemistry↗

bioRxiv2020

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The formation of cellular microtubule networks is regulated by the{gamma} -tubulin ring complex ({gamma}-TuRC). This [~]2.3 MDa assembly of >31 proteins includes{gamma} -tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge". The challenge of reconstituting the{gamma} -TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human{gamma} -TuRC ({gamma}-TuRC-GFP), a [~]35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex,{gamma} -TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that{gamma} -TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native{gamma} -TuRCs. Electron microscopy reveals that{gamma} -TuRC-GFP resembles the native{gamma} -TuRC architecture, while{gamma} -TuRCmini-GFP adopts a partial cone shape presenting only 8-10{gamma} -tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the{gamma} -TuRC.

biochemistry↗

bioRxiv2020

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

biophysics↗

bioRxiv2019

CLASP mediates microtubule repair by promoting tubulin incorporation into damaged lattices

Microtubule network plays a key role in cell division, motility and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can either be induced by mechanical stress or occur spontaneously during polymerization [2-4]. Tubulin incorporation can restore microtubule integrity; moreover, \"islands\" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3-7]. Microtubule repair occurs in vitro in the presence of tubulin alone [2-4, 8]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASP is an interesting candidate for microtubule repair, because it induces microtubule nucleation, stimulates rescue and suppresses catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [9-16]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2 indeed stimulates microtubule lattice repair. CLASP2 promoted tubulin incorporation into damaged lattice sites thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic flow-induced bending. A single CLASP2 domain, TOG2, which suppresses catastrophes when tethered to microtubules, was sufficient to stimulate microtubule repair, indicating that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.

cell biology↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗

bioRxiv2018

Exocyst subcomplex functions in autophagosome biogenesis by regulating Atg9 trafficking

During autophagy, double membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of Exocyst- an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst which are dispensable for autophagic function do not associate with the autophagy specific subcomplex of exocyst.

cell biology↗