The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin(-/-) mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.
Ultrashort echo time (UTE) imaging is a well-known technique used in medical MRI, however, the implementation of the sequence remains non-trivial. This paper introduces UTE for non-medical applications and outlines a method for the implementation of UTE to enable accurate slice selection and short acquisition times. Slice selection in UTE requires fast, accurate switching of the gradient and r.f. pulses. Here a gradient "pre-equalization" technique is used to optimize the gradient switching and achieve an effective echo time of 10μs. In order to minimize the echo time, k-space is sampled radially. A compressed sensing approach is used to minimize the total acquisition time. Using the corrections for slice selection and acquisition along with novel image reconstruction techniques, UTE is shown to be a viable method to study samples of cork and rubber with a shorter signal lifetime than can typically be measured. Further, the compressed sensing image reconstruction algorithm is shown to provide accurate images of the samples with as little as 12.5% of the full k-space data set, potentially permitting real time imaging of short T2(*) materials.
Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca2+ ions generates gels of different bulk homogeneity dependent on the alginate structure. Shorter spin-spin T2 magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.
SUMMARY Bioenergy sorghum is a highly productive drought tolerant C4 grass that accumulates 80% of its harvestable biomass in approximately 4 m length stems. Stem internode growth is regulated by development, shading, and hormones that modulate cell proliferation in intercalary meristems (IMs). In this study, sorghum stem IMs were localized above the pulvinus at the base of elongating internodes using magnetic resonance imaging, microscopy, and transcriptome analysis. A change in cell morphology/organization occurred at the junction between the pulvinus and internode where LATERAL ORGAN BOUNDARIES (SbLOB), a boundary layer gene, was expressed. Inactivation of an AGCVIII kinase in DDYM (dw2) resulted in decreased SbLOB expression, disrupted IM localization, and reduced internode cell proliferation. Transcriptome analysis identified approximately 1000 genes involved in cell proliferation, hormone signaling, and other functions selectively upregulated in the IM compared with a non‐meristematic stem tissue. This cohort of genes is expressed in apical dome stem tissues before localization of the IM at the base of elongating internodes. Gene regulatory network analysis identified connections between genes involved in hormone signaling and cell proliferation. The results indicate that gibberellic acid induces accumulation of growth regulatory factors (GRFs) known to interact with ANGUSTIFOLIA (SbAN3), a master regulator of cell proliferation. GRF:AN3 was predicted to induce SbARF3/ETT expression and regulate SbAN3 expression in an auxin‐dependent manner. GRFs and ARFs regulate genes involved in cytokinin and brassinosteroid signaling and cell proliferation. The results provide a molecular framework for understanding how hormone signaling regulates the expression of genes involved in cell proliferation in the stem IM.
The formation of heterogeneous structures in biopolymer gels is of current interest for biomedical applications and is of fundamental interest to understanding the molecular level origins of structures generated from disordered solutions by reactions. The cation-mediated physical gelation of alginate by calcium and copper is analyzed using magnetic resonance measurements of spatially resolved molecular dynamics during gel front propagation. Relaxation time and pulse-field gradient methods are applied to determine the impact of ion front motion on molecular translational dynamics. The formation of capillaries in alginate copper gels is correlated to changes in translational dynamics.
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