Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Impaired immunity is a fundamental obstacle to successful allogeneic hematopoietic cell transplantation. Mature graft T cells are thought to provide protection from infections early after transplantation, but can cause life-threatening graft-vs.-host disease. Human CMV is a major pathogen after transplantation. We studied reactivity against the mouse homologue, murine CMV (MCMV), in lethally irradiated mice given allogeneic purified hematopoietic stem cells (HSCs) or HSCs supplemented with T cells or T-cell subsets. Unexpectedly, recipients of purified HSCs mounted superior antiviral responses compared with recipients of HSC plus unselected bulk T cells. Furthermore, supplementation of purified HSC grafts with CD8 + memory or MCMV-specific T cells resulted in enhanced antiviral reactivity. Posttransplantation lymphopenia promoted massive expansion of MCMV-specific T cells when no competing donor T cells were present. In recipients of pure HSCs, naive and memory T cells and innate lymphoid cell populations developed. In contrast, the lymphoid pool in recipients of bulk T cells was dominated by effector memory cells. These studies show that pure HSC transplantations allow superior protective immunity against a viral pathogen compared with unselected mature T cells. This reductionist transplant model reveals the impact of graft composition on regeneration of host, newly generated, and mature transferred T cells, and underscores the deleterious effects of bulk donor T cells. Our findings lead us to conclude that grafts composed of purified HSCs provide an optimal platform for in vivo expansion of selected antigen-specific cells while allowing the reconstitution of a naive T-cell pool.graft-vs.-host reactions | hematopoietic stem cell transplantation | immune reconstitution | lymphopenia induced proliferation | M45-tetramer

As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.

Many biological machines function in discrete steps, and detection of such steps can provide insight into the machines' dynamics. It is therefore crucial to develop an automated method to detect steps, and determine how its success is impaired by the significant noise usually present. A number of step detection methods have been used in previous studies, but their robustness and relative success rate have not been evaluated. Here, we compare the performance of four step detection methods on artificial benchmark data (simulating different data acquisition and stepping rates, as well as varying amounts of Gaussian noise). For each of the methods we investigate how to optimize performance both via parameter selection and via prefiltering of the data. While our analysis reveals that many of the tested methods have similar performance when optimized, we find that the method based on a chi-squared optimization procedure is simplest to optimize, and has excellent temporal resolution. Finally, we apply these step detection methods to the question of observed step sizes for cargoes moved by multiple kinesin motors in vitro. We conclude there is strong evidence for sub-8-nm steps of the cargo's center of mass in our multiple motor records.

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