Major, J. | Search bioRxiv

bioRxiv2019

The Kinesin-5 Tail Domain Directly Modulates the Mechanochemical Cycle of the Motor for Anti-Parallel Microtubule Sliding

Kinesin-5 motors organize mitotic spindles by sliding apart anti-parallel microtubules. They are homotetramers composed of two antiparallel dimers placing orthogonal motor and tail domains at opposite ends of a bipolar minifilament. Here, we describe a regulatory mechanism, involving direct binding of the tail to motor domain and reveal its fundamental role in microtubule sliding motility. Biochemical analyses reveal that the tail down-regulates microtubule-activated ATP hydrolysis by specifically engaging the motor in the nucleotide-free or ADP-bound states. Cryo-EM structures reveal that the tail stabilizes a unique conformation of the motor N-terminal subdomain opening its active site. Full-length kinesin-5 motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering along microtubules. The tail is critical for motors to zipper together two microtubules by generating substantial forces within sliding zones. The tail domain is essential for kinesin-5 mitotic spindle localization in vivo, which becomes severely reduced when the tail is deleted. Our studies suggest a revised microtubule-sliding model, in which tail domains directly engage motor domains at both ends of kinesin-5 homotetramers enhancing stability of the dual microtubule-bound states leading to slow motility yet high force production.

biophysics↗

bioRxiv2019

Pan-viral protection against arboviruses by targeting inoculation site-based skin macrophages

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The large number of genetically-distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made developing virus-specific anti-viral medicines challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies at this site. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical innate immune agonist significantly suppressed both the local and subsequent systemic course of infection and improved clinical outcome in mice to infection with a variety of arboviruses from the Alphavirus, Flavivirus and Orthobunyavirus genuses. In the absence of treatment, anti-viral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, targeting the more populous skin-resident macrophages with an immune agonist elicited protective responses in key cellular targets of virus that otherwise replicated virus to high levels. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have shown that it is possible to improve outcome to infection by targeting pathways activated at the site of inoculation, and thereby identified a putative new strategy for limiting disease following infection with a variety of genetically-distinct arboviruses.\n\nOne-sentence summaryWe demonstrate that activation of innate immune responses to arbovirus at the mosquito bite is a limiting factor for preventing efficient systemic dissemination of virus and that therapeutic targeting of skin-resident macrophages can have defining inhibitory effects on the later systemic course.

immunology↗