Browsing by Author "Roy, Pallabi"

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    Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin
    (Elsevier, 2021-03) McGrath, Jamis; Tung, Chun-Yu; Liao, Xiayi; Belyantseva, Inna A.; Roy, Pallabi; Chakraborty, Oisorjo; Li, Jinan; Berbari, Nicolas F.; Faaborg-Andersen, Christian C.; Barzik, Melanie; Bird, Jonathan E.; Zhao, Bo; Balakrishnan, Lata; Friedman, Thomas B.; Perrin, Benjamin J.; Biology, School of Science
    Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.
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    Essential nucleotide- and protein-dependent functions of Actb/β-actin
    (National Academy of Sciences, 2018-07-31) Patrinostro, Xiaobai; Roy, Pallabi; Lindsay, Angus; Chamberlain, Christopher M.; Sundby, Lauren J.; Starker, Colby G.; Voytas, Daniel F.; Ervasti, James M.; Perrin, Benjamin J.; Biochemistry and Molecular Biology, School of Medicine
    The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.
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    Molecular cloning of the soybean phototropins
    (2014) Roy, Pallabi; Watson, John C., 1953-; Randall, Stephen Karl, 1953-; Anderson, Gregory G.; Atkinson, Simon
    The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.
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    The stable actin core of mechanosensory stereocilia features continuous turnover of actin cross-linkers
    (American Society for Cell Biology, 2018-08-01) Roy, Pallabi; Perrin, Benjamin J.; Biology, School of Science
    Stereocilia are mechanosensitive protrusions on the surfaces of sensory hair cells in the inner ear that detect sound, gravity, and head movement. Their cores are composed of parallel actin filaments that are cross-linked and stabilized by several actin-binding proteins, including fascin-2, plastin-1, espin, and XIRP2. The actin filaments are the most stable known, with actin turnover primarily occurring at the stereocilia tips. While stereocilia actin dynamics has been well studied, little is known about the behavior of the actin cross-linking proteins, which are the most abundant type of protein in stereocilia after actin and are critical for stereocilia morphogenesis and maintenance. Here, we developed a novel transgenic mouse to monitor EGFP-fascin-2 incorporation . In contrast to actin, EGFP-fascin-2 readily enters the stereocilia core. We also compared the effect of EGFP-fascin-2 expression on developing and mature stereocilia. When it was induced during hair cell development, we observed increases in both stereocilia length and width. Interestingly, stereocilia size was not affected when EGFP-fascin-2 was induced in adult stereocilia. Regardless of the time of induction, EGFP-fascin-2 displaced both espin and plastin-1 from stereocilia. Altering the actin cross-linker composition, even as the actin filaments exhibit little to no turnover, provides a mechanism for ongoing remodeling and repair important for stereocilia homeostasis.
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    Stereocilia Morphogenesis and Maintenance is Dependent on the Dynamics of Actin Cytoskeletal Proteins
    (2019-05) Roy, Pallabi; Perrin, Benjamin J.; Atkinson, Simon J.; Belecky-Adams, Teri; Hashino, Eri; Baucum II, Anthony J.
    Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and maintenance of stereocilia.
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    Stereocilia morphogenesis and maintenance through regulation of actin stability
    (Elsevier, 2017-05) McGrath, Jamis; Roy, Pallabi; Perrin, Benjamin J.; Biology, School of Science
    Stereocilia are actin-based protrusions on auditory and vestibular sensory cells that are required for hearing and balance. They convert physical force from sound, head movement or gravity into an electrical signal, a process that is called mechanoelectrical transduction. This function depends on the ability of sensory cells to grow stereocilia of defined lengths. These protrusions form a bundle with a highly precise geometry that is required to detect nanoscale movements encountered in the inner ear. Congenital or progressive stereocilia degeneration causes hearing loss. Thus, understanding stereocilia hair bundle structure, development, and maintenance is pivotal to understanding the pathogenesis of deafness. Stereocilia cores are made from a tightly packed array of parallel, crosslinked actin filaments, the length and stability of which are regulated in part by myosin motors, actin crosslinkers and capping proteins. This review aims to describe stereocilia actin regulation in the context of an emerging "tip turnover" model where actin assembles and disassembles at stereocilia tips while the remainder of the core is exceptionally stable.