The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

Anillin interacts with microtubules and is part of the astral pathway that defines cortical domains.

Cytokinesis occurs by the ingression of an actomyosin ring that separates the cell into two daughter cells. The mitotic spindle, comprising astral and central spindle microtubules, couples contractile ring ingression with DNA segregation. Cues from the central spindle activate RhoA, the upstream regulator of the contractile ring. However, additional cues from the astral microtubules also reinforce the localization of active RhoA. Using human cells, we show that astral and central spindle microtubules independently control the localization of contractile proteins during cytokinesis. Astral microtubules restrict the accumulation and localization of contractile proteins during mitosis, whereas the central spindle forms a discrete ring by directing RhoA activation in the equatorial plane. Anillin stabilizes the contractile ring during cytokinesis. We show that human anillin interacts with astral microtubules and that this interaction is competed by the cortical recruitment of anillin by active RhoA. Anillin restricts the localization of myosin to the equatorial cortex and that of NuMA (part of the microtubule-tethering complex that regulates spindle position) to the polar cortex. The sequestration of anillin by astral microtubules might alter the organization of cortical proteins to polarize cells for cytokinesis.

Optogenetic control of kinesin-1, -2, -3 and dynein reveals their specific roles in vesicular transport.

Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.

Reconstitution of Organelle Transport Along Microtubules In Vitro.

In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.

Importin binding mediates the intramolecular regulation of anillin during cytokinesis.

Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains a nuclear localization signal (NLS) that binds to importin and is required for its function during cytokinesis. Here we reveal the mechanism whereby importin binding favors a conformation required for anillin's recruitment to the equatorial cortex. Active RhoA binds to the RhoA-binding domain causing an increase in accessibility of the nearby C2 domain containing the NLS. Importin binding subsequently stabilizes a conformation that favors interactions for cortical recruitment. In addition to revealing a novel mechanism for the importin-mediated regulation of a cortical protein, we also show how importin binding positively regulates protein function.

Active Ran regulates anillin function during cytokinesis.

Cytokinesis cleaves a cell into two daughters at the end of mitosis, and must be spatially coordinated with chromosome segregation to prevent aneuploidy. The dogma is that the mitotic spindle governs the assembly and constriction of an actomyosin ring. Here, we reveal a function for active Ran in spatially restricting the ring. Our model is that during anaphase, "free" importins, whose gradient inversely correlates with active Ran and chromatin position, function as a molecular ruler for the recruitment and localization of anillin, a contractile protein and a crucial regulator of cytokinesis. We found that decreasing Ran-GTP levels or tethering active Ran to the equatorial membrane affects anillin's localization and causes cytokinesis phenotypes. Anillin contains a conserved nuclear localization signal (NLS) at its C-terminus that binds to importin-ß and is required for cortical polarity and cytokinesis. Mutating the NLS decreases anillin's cortical affinity, causing it to be more dominantly regulated by microtubules. Anillin contains a RhoA-GTP binding domain, which autoinhibits the NLS and the neighboring microtubule-binding domain, and RhoA-GTP binding may relieve this inhibition during mitosis. Retention of the C-terminal NLS in anillin homologues suggests that this is a conserved mechanism for controlling anillin function.

Comparative study on cellular entry of incinerated ancient gold particles (Swarna Bhasma) and chemically synthesized gold particles.

Gold nanoparticles (AuNPs) are used for a number of imaging and therapeutic applications in east and western part of the world. For thousands of years, the traditional Indian Ayurvedic approach to healing involves the use of incinerated gold ash, prepared with a variety of plant extracts and minerals depending on the region. Here, we describe the characterization of incinerated gold particles (IAuPs) in HeLa (human cells derived from cervical cancer) and HFF-1 (human foreskin fibroblast cells) in comparison to synthesized citrate-capped gold nanoparticles (AuNPs). We found that while individual IAuP crystallites are around 60 nm in size, they form large aggregates with a mean diameter of 4711.7 nm, some of which can enter cells. Fewer cells appeared to have IAuPs compared to AuNPs, although neither type of particle was toxic to cells. Imaging studies revealed that IAuPs were in vesicles, cytosol, or in the nucleus. We found that their nuclear accumulation likely occurred after nuclear envelope breakdown during cell division. We also found that larger IAuPs entered cells via macropinocytosis, while smaller particles entered via clathrin-dependent receptor-mediated endocytosis.