Imaging the dynamic recruitment of monocytes to the blood-brain barrier and specific brain regions during Toxoplasma gondii infection.

Brain infection by the parasite Toxoplasma gondii in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling T. gondii However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2+Ly6Chi) and patrolling (CX3CR1+Ly6Clo) monocytes into the blood and brain during T. gondii infection of C57BL/6J and CCR2RFP/+CX3CR1GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood-brain barrier (BBB) within 2 wk of T. gondii infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains using iDISCO+ and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of T. gondii and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of T. gondii clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., Nature 445:168-176 (2007)] indicated a consistent pattern of monocyte infiltration during T. gondii infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during T. gondii CNS infection.

Microtubule C-Terminal Tails Can Change Characteristics of Motor Force Production.

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties--if at all--is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin-1, and also a mitotic kinesin likely optimized for group function, kinesin-5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin-1 functioned similarly on the two sets of MTs--in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin-1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin-5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for kinesin-5 on the cancer-cell MTs, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar.

Load-induced enhancement of Dynein force production by LIS1-NudE in vivo and in vitro.

Most sub-cellular cargos are transported along microtubules by kinesin and dynein molecular motors, but how transport is regulated is not well understood. It is unknown whether local control is possible, for example, by changes in specific cargo-associated motor behaviour to react to impediments. Here we discover that microtubule-associated lipid droplets (LDs) in COS1 cells respond to an optical trap with a remarkable enhancement in sustained force production. This effect is observed only for microtubule minus-end-moving LDs. It is specifically blocked by RNAi for the cytoplasmic dynein regulators LIS1 and NudE/L (Nde1/Ndel1), but not for the dynactin p150(Glued) subunit. It can be completely replicated using cell-free preparations of purified LDs, where duration of LD force production is more than doubled. These results identify a novel, intrinsic, cargo-associated mechanism for dynein-mediated force adaptation, which should markedly improve the ability of motor-driven cargoes to overcome subcellular obstacles.