Microtubule capture by mitotic kinesin centromere protein E (CENP-E).

Centromere protein E, CENP-E, is a kinetochore-associated kinesin-7 that establishes the microtubule-chromosome linkage and transports monooriented chromosomes to the spindle equator along kinetochore fibers of already bioriented chromosomes. As a processive kinesin, CENP-E uses a hand-over-hand mechanism, yet a number of studies suggest that CENP-E exhibits mechanistic differences from other processive kinesins that may be important for its role in chromosome congression. The results reported here show that association of CENP-E with the microtubule is unusually slow at 0.08 µM(-1) s(-1) followed by slow ADP release at 0.9 s(-1). ATP binding and hydrolysis are fast with motor dissociation from the microtubule at 1.4 s(-1), suggesting that CENP-E head detachment from the microtubule, possibly controlled by phosphate release, determines the rate of stepping during a processive run because the rate of microtubule gliding corresponds to 1.4 steps/s. We hypothesize that the unusually slow CENP-E microtubule association step favors CENP-E binding of stable microtubules over dynamic ones, a mechanism that would bias CENP-E binding to kinetochore fibers.

Aerosol delivery of SARS-CoV-2 human monoclonal antibodies in macaques limits viral replication and lung pathology.

Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.

Mitotic kinesin CENP-E promotes microtubule plus-end elongation.

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 µm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,ß-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

A Cellular Networking Model Involving Interactions Among Glycosyl-Phosphatidylinositol (GPI)-Anchored Plasma Membrane Arabinogalactan Proteins (AGPs), Microtubules and F-actin in Tobacco BY-2 Cells.

Arabinogalactan-proteins (AGPs) are perhaps the most abundantly expressed set of proteins at the plant cell surface and play probable roles in cellular architecture and signaling. Although considerable progress has been made to understand the role of AGPs in plant growth and development, their exact functional roles and the molecular mechanisms underlying their interactions with either intra- or extra-cellular molecules are unknown. These unknown interactions were addressed in a recent research article in Plant Physiology. This study reported molecular interactions between AGPs and the cytoskeleton [microtubules, (MTs) and F-actin] in tobacco BY-2 cells. Here in this addendum, a summary of this recent publication and additional perspectives are presented. As reported, perturbation studies were conducted in tobacco BY-2 cells to analyze the effects of an AGP inhibitor (beta-Yariv reagent) on the organization of microtubules [labeled by GFP-MBD (green fluorescent protein-microtubule binding domain)] and F-actin (labeled by rhodamine-phalloidin) and conversely to analyze the effects of a microtubule inhibitor (amiprophosmethyl) and an F-actin inhibitor (cytochalasin-D) on the localization of GPI-anchored GFP-LeAGP-1. These studies implicate a role for GPI-anchored LeAGP-1 in mediating a cell wall-plasma membrane-cytoskeleton connection.

A lysine-rich arabinogalactan protein in Arabidopsis is essential for plant growth and development, including cell division and expansion.

Arabinogalactan proteins (AGPs), a family of hydroxyproline-rich glycoproteins, occur throughout the plant kingdom. The lysine-rich classical AGP subfamily in Arabidopsis consists of three members, AtAGP17, 18 and 19. In this study, AtAGP19 was examined in terms of its gene expression pattern and function. AtAGP19 mRNA was abundant in stems, with moderate levels in flowers and roots and low levels in leaves. AtAGP19 promoter-controlled GUS activity was high in the vasculature of leaves, roots, stems and flowers, as well as styles and siliques. A null T-DNA knockout mutant of AtAGP19 was obtained and compared to wild-type (WT) plants. The atagp19 mutant had: (i) smaller, rounder and flatter rosette leaves, (ii) lighter-green leaves containing less chlorophyll, (iii) delayed growth, (iv) shorter hypocotyls and inflorescence stems, and (v) fewer siliques and less seed production. Several abnormalities in cell size, number, shape and packing were also observed in the mutant. Complementation of this pleiotropic mutant with the WT AtAGP19 gene restored the WT phenotypes and confirmed that AtAGP19 functions in various aspects of plant growth and development, including cell division and expansion, leaf development and reproduction.

Molecular interactions of arabinogalactan proteins with cortical microtubules and F-actin in Bright Yellow-2 tobacco cultured cells.

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.