A cell’s mechanical properties are important in determining its adhesion, migration, and response to the mechanical properties of its microenvironment and may help explain behavioral differences between normal and cancerous cells. Using fluorescently labeled peroxisomes as microrheological probes, the interior mechanical properties of normal breast cells were compared to a metastatic breast cell line, MDA-MB-231. To estimate the mechanical properties of cell cytoplasms from the motions of their peroxisomes, it was necessary to reduce the contribution of active cytoskeletal motions to peroxisome motion. This was done by treating the cells with blebbistatin, to inhibit myosin II, or with sodium azide and 2-deoxy-D-glucose, to reduce intracellular ATP. Using either treatment, the peroxisomes exhibited normal diffusion or subdiffusion, and their mean squared displacements (MSDs) showed that the MDA-MB-231 cells were significantly softer than normal cells. For these two cell types, peroxisome MSDs in treated and untreated cells converged at high frequencies, indicating that cytoskeletal structure was not altered by the drug treatment. The MSDs from ATP-depleted cells were analyzed by the generalized Stokes–Einstein relation to estimate the interior viscoelastic modulus G* and its components, the elastic shear modulus G′ and viscous shear modulus G″, at angular frequencies between 0.126 and 628rad/s. These moduli are the material coefficients that enter into stress–strain relations and relaxation times in quantitative mechanical models such as the poroelastic model of the interior regions of cancerous and non-cancerous cells.

Abstract. We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in the nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340±30 nm, which simultaneously photoactivate a 7x7 matrix pattern of GFP-labeled histones, with spots 1.70 µm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals DNA damage-dependent decrease in chromatin diffusion. The DOE instrumentation can easily and cheaply be implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in non-adherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

Salmonella contamination associated with market fresh tomatoes has been problematic for the industry and consumers. A number of outbreaks have occurred, and dollar losses for the industry, including indirect collateral impact to agriculturally connected communities, have run into the hundreds of millions of dollars. This review covers these issues and an array of problems and potential solutions surrounding Salmonella contamination in tomatoes. Some other areas discussed include (i) the use of case-control studies and DNA fingerprinting to identify sources of contamination, (ii) the predilection for contamination based on Salmonella serovar and tomato cultivar, (iii) internalization, survival, and growth of Salmonella in or on tomatoes and the tomato plant, in biofilms, and in niches ancillary to tomato production and processing, (iv) the prevalence of Salmonella in tomatoes, especially in endogenous regions, and potential sources of contamination, and (v) effective and experimental means of decontaminating Salmonella from the surface and stem scar regions of the tomato. Future research should be directed in many of the areas discussed in this review, including determining and eliminating sources of contamination and targeting regions of the country where Salmonella is endemic and contamination is most likely to occur. Agriculturalists, horticulturalists, microbiologists, and epidemiologists may make the largest impact by working together to solve other unanswered questions regarding tomatoes and Salmonella contamination.

The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCENucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions. N ucleocapsids of negative-strand RNA viruses must be transported from their sites of assembly in the cytoplasm to sites of virus budding from host membranes (1). For example, the nucleocapsids of vesicular stomatitis virus (VSV) behave as random coils with a hydrodynamic radius of approximately 90 nm (2), which is too large to diffuse through the cytoplasm in the time required for virus assembly (3). Transport of nucleocapsids to the membrane after assembly in the cytoplasm has been proposed to occur primarily along microtubules (4). The goal of the experiments presented here was to further test mechanisms of nucleocapsid transport by evaluating both microtubule-dependent and actin-dependent transport using recently developed analytical tools.Actin filaments and microtubules have a general orientation in which the growing (plus) end is oriented toward the cell periphery and the minus end is oriented toward the center of the cell (5). Assembly of microtubules is usually...

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We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340  ±  30  nm, which simultaneously photoactivate a 7  ×  7 matrix pattern of GFP-labeled histones, with spots 1.70  μm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

Many organelles and vesicles in live cells move in a start–stop manner when observed for ~10 s by optical microscopy. Changes in velocity and directional persistence of such particles are a potentially rich source of insight into the mechanisms leading to the start and stop states. Unbiased assessment of the most probable number of states, the properties of each state, and the most probable state for the particle at each moment can be accomplished by variational Bayesian methods combined with a hidden Markov model and a Gaussian mixture model. Our track analysis method, “vbTRACK”, applied this combination of methods to particle velocity v or changes in the direction of travel evaluated from simulated tracks and from tracks of peroxisomes in live cells. When tested with numerical data, vbTRACK reliably determined the number of states, the mean and variance of the velocity or the direction of travel for each state, and the most probable state during each frame. When applied to the tracks of peroxisomes in live cells, some tracks separated into two states, one with high velocity and directionality, the other approximately Brownian. Other tracks of particles in live cells separated into several diffusive states with distinct diffusion constants.