Fluorescence Correlation Spectroscopy analysis of segmental dynamics in Actin filaments

We adapt Fluorescence Correlation spectroscopy (FCS) formalism to the studies of the dynamics of semi-flexible polymers and derive expressions relating FCS correlation function to the longitudinal and transverse mean square displacements of polymer segments. We use the derived expressions to measure the dynamics of actin filaments in two experimental situations: filaments labeled at distinct positions and homogeneously labeled filaments. Both approaches give consistent results and allow to measure the temporal dependence of the segmental mean-square displacement (MSD) over almost five decades in time, from ~0.04ms to 2s. These noninvasive measurements allow for a detailed quantitative comparison of the experimental data to the current theories of semi-flexible polymer dynamics. Good quantitative agreement is found between the experimental results and theories explicitly accounting for the hydrodynamic interactions between polymer segments

Rice microtubule‐associated protein IQ67‐DOMAIN14 regulates rice grain shape by modulating microtubule cytoskeleton dynamics

Cortical microtubule (MT) arrays play a critical role in plant cell shape determination by defining the direction of cell expansion. As plants continuously adapt to ever‐changing environmental conditions, multiple environmental and developmental inputs need to be translated into changes of the MT cytoskeleton. Here, we identify and functionally characterize an auxin‐inducible and MT‐localized protein OsIQ67‐DOMAIN14 (OsIQD14), which is highly expressed in rice seed hull cells. We show that while deficiency of OsIQD14 results in short and wide seeds and increases overall yield, overexpression leads to narrow and long seeds, caused by changed MT alignment. We further show that OsIQD14‐mediated MT reordering is regulated by specifically affecting MT dynamics, and ectopic expression of OsIQD14 in Arabidopsis could change the cell shape both in pavement cells and hypocotyl cells. Additionally, OsIQD14 activity is tightly controlled by calmodulin proteins, providing an alternative way to modify the OsIQD14 activity. Our results indicate that OsIQD14 acts as a key factor in regulating MT rearrangements in rice hull cells and hence the grain shape, and allows effective local cell shape manipulation to improve the rice yield trait

Holocentric Chromosomes of Luzula elegans Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability. Copyright (C) 2011 S. Karger AG, Base

Super-resolution and super-localization microscopy: a novel tool for imaging chemical and biological processes

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes. The obtained spatial resolution for plant cell imaging is not yet as good as that achieved in mammalian cell imaging. Numerous technical challenges, including the generally high fluorescence background due to significant autofluorescence of endogenous components, and the presence of the cell wall (\u3e 250 nm thickness) limit the potential of super-resolution imaging in studying the cellular processes in plants. Here variable-angle epi-fluorescence microscopy (VAEM) was combined with localization based super-resolution imaging, direct stochastic optical reconstruction microscopy (dSTORM), to demonstrate imaging of cortical microtubule (CMT) network in the Arabidopsis thaliana root cells with 20 – 40 nm spatial resolution for the first time. With such high spatial resolution, the subcellular organizations of CMTs within single cells, and different cells in many regions along the root, were analyzed quantitatively. Nearly all of these technical advances in super-localization and super-resolution microscopy imaging were originally developed for biological studies. More recently, however, efforts in super-resolution chemical imaging started to gain momentum. New discoveries that were previously unattainable with conventional diffraction-limited techniques have been made, such as a) super-resolution mapping of catalytic reactions on single nanocatalysts and b) mechanistic insight into protein ion-exchange adsorptive separations. Furthermore, single molecules and single particles were localized with nanometer precision for resolving the dynamic behavior of single molecules in porous materials. This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM). To fully understand the working mechanisms of biological processes such as stepping of motor proteins requires resolving both the translational movement and the rotational motions of biological molecules or molecular complexes. Nanoparticle optical probes have been widely used to study biological processes such as membrane diffusion, endocytosis, and so on. The greatly enhanced absorption and scattering cross sections at the surface plasmon resonance (SPR) wavelength make nanoparticles an ideal probe for high precision tracking. Furthermore, gold nanorods (AuNRs) were used for resolving orientation changes in all three dimensions. The translational and rotational motions of AuNRs in glycerol solutions were tracked with fast imaging rate up to 500 frames per second (fps) in reflected light sheet microscopy (RLSM). The effect of imaging rates on resolving details of single AuNR motions was studied

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates

Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots

Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains

The development of optical microscopy techniques for the advancement of single-particle studies

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called non-blinking quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces

Using Microscopy to Examine Mechanisms of Cell Division

The accurate segregation of chromosomes during cell division requires the assembly of a microtubule-based mitotic spindle. In the research presented here, I examined two aspects of mitotic spindle morphogenesis. First, I looked at how chromosomes attach to microtubules in mitosis. The capture of microtubule plus-ends by kinetochores (the site of chromosome-microtubule attachment) has been described previously. However, recent data suggests that another mechanism must also contribute to this essential process. Here, with the aid of real-time confocal microscopy, I present direct evidence for an additional chromosome-spindle attachment mechanism in which the minus-ends of microtubules attached to chromosomes are captured by spindle microtubules and incorporated into the spindle. I also show that this process depends on the microtubule cross-linking MAP (microtubule associated protein), NuMA. Second, I examined the molecular basis of bipolar spindle formation: in particular, contributions of the non-motor MAP TPX2 to spindle structure and function in mammalian cells. My live cell recordings revealed that in cells depleted of TPX2, multipolar spindles formed by progressive spindle pole fragmentation. Consistent with structural role for TPX2 in the spindle, I found that while normal bipolar spindles resistant to collapse to monopolar spindles after inhibition of the mitotic motor Eg5, multipolar spindles that formed in the absence of TPX2 were not. In addition, I found that microtubule nucleation and growth during spindle assembly is not dependent on TPX2, and that cells form robust kinetochore microtubule bundles after TPX2 knockdown. These data do not suggest a direct role of TPX2 in the establishment of chromosome-microtubule attachments. However, my results shed new light on the spindle defects in cells without T P X 2 and confirm an important role for T P X 2 in the structural stability of the mitotic spindle

Reconfigurable Flows and Defect Landscape of Confined Active Nematics

Using novel micro-printing techniques, we develop a versatile experimental setup that allows us to study how lateral confinement tames the active flows and defect properties of the microtubule/kinesin active nematic system. We demonstrate that the active length scale that determines the self-organization of this system in unconstrained geometries loses its relevance under strong lateral confinement. Dramatic transitions are observed from chaotic to vortex lattices and defect-free unidirectional flows. Defects, which determine the active flow behavior, are created and annihilated on the channel walls rather than in the bulk, and acquire a strong orientational order in narrow channels. Their nucleation is governed by an instability whose wavelength is effectively screened by the channel width. All these results are recovered in simulations, and the comparison highlights the role of boundary conditions