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Ram Dixit

Publications and source records attributed to Ram Dixit.

7 recordsLinked to original sources
PubMed2006

Using intrinsically fluorescent proteins for plant cell imaging.

The intrinsically fluorescent proteins (IFPs), such as the green, cyan and yellow fluorescent proteins, have revolutionized how we can image the dynamics of cellular events. Intrinsically fluorescent proteins have been used as reporter genes to monitor transcriptional regulation, as targeted markers for organelles and subcellular structures, in fusion proteins to directly observe protein motility and dynamics, and in sensors designed to show changes in cellular environments ranging from pH to protein kinase activity. The IFPs hold tremendous potential to reveal the dynamic processes that underlie plant cell function; however, as with all technology there are artifacts and pitfalls inherent in their use. In this review, we highlight some of the practical issues in using IFPs for live cell imaging. These include choice of the appropriate IFP, dealing with autofluorescence, photobleaching and phototoxicity, and application of approaches such as fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM) and fluorescence recovery after photobleaching (FRAP) to gain high-resolution data about protein dynamics within the cell. We also discuss some of the more common artifacts associated with these fluorescence imaging approaches and suggest controls that should help both spot these problems and suggest their solutions.

Fluorescence Resonance Energy Transfer↗
PubMed2005

Establishment of polarity during organization of the acentrosomal plant cortical microtubule array.

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.

Amino Acid Sequence↗
PubMed2004

Arabidopsis WPP-domain proteins are developmentally associated with the nuclear envelope and promote cell division.

The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference-based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity.

Arabidopsis↗
PubMed2004

Encounters between dynamic cortical microtubules promote ordering of the cortical array through angle-dependent modifications of microtubule behavior.

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis alpha-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome of such encounters depends on the angle at which they occur: steep-angle collisions are characterized by approximately sevenfold shorter microtubule contact times compared with shallow-angle encounters, and steep-angle collisions are twice as likely to result in microtubule depolymerization. Hence, steep-angle collisions promote microtubule destabilization, whereas shallow-angle encounters promote both microtubule stabilization and coalignment. Monte Carlo modeling of the behavior of simulated microtubules, according to the observed behavior of transverse and longitudinally oriented cortical microtubules in cells, reveals that these simple rules for intermicrotubule interactions are necessary and sufficient to facilitate the self-organization of dynamic microtubules into a parallel configuration.

Bacterial Proteins↗
PubMed2003

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.

Cells, Cultured↗
PubMed2002

Golgi secretion is not required for marking the preprophase band site in cultured tobacco cells.

The preprophase band predicts the future cell division site. However, the mechanism of how a transient preprophase band fulfils this function is unknown. We have investigated the possibility that Golgi secretion might be involved in marking the preprophase band site. Observations on living BY-2 cells labeled for microtubules and Golgi stacks indicated an increased Golgi stack frequency at the preprophase band site. However, inhibition of Golgi secretion by brefeldin A during preprophase band formation did not prevent accurate phragmoplast fusion, and subsequent cell plate formation, at the preprophase band site. The results show that Golgi secretion does not mark the preprophase band site and thus does not play an active role in determination of the cell division site.

Amino Acid Sequence↗