Ram Dixit

Differential Regulation of Dynein and Kinesin Motor Proteins by Tau

Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.

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 α-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.

Microtubule plus-end tracking by CLIP-170 requires EB1

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TIPs) localize to and track with growing microtubule plus-ends in the cell. +TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our single-molecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170–EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5′-[α,β-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.

A Switch in Retrograde Signaling from Survival to Stress in Rapid-Onset Neurodegeneration

Retrograde axonal transport of cellular signals driven by dynein is vital for neuronal survival. Mouse models with defects in the retrograde transport machinery, including the Loa mouse (point mutation in dynein) and the Tgdynamitin mouse (overexpression of dynamitin), exhibit mild neurodegenerative disease. Transport defects have also been observed in more rapidly progressive neurodegeneration, such as that observed in the SOD1G93A transgenic mouse model for familial amyotrophic lateral sclerosis (ALS). Here, we test the hypothesis that alterations in retrograde signaling lead to neurodegeneration. In vivo, in vitro, and live-cell imaging motility assays show misregulation of transport and inhibition of retrograde signaling in the SOD1G93A model. However, similar inhibition is also seen in the Loa and Tgdynamitin mouse models. Thus, slowing of retrograde signaling leads only to mild degeneration and cannot explain ALS etiology. To further pursue this question, we used a proteomics approach to investigate dynein-associated retrograde signaling. These data indicate a significant decrease in retrograde survival factors, including P-Trk (phospho-Trk) and P-Erk1/2, and an increase in retrograde stress factor signaling, including P-JNK (phosphorylated c-Jun N-terminal kinase), caspase-8, and p75NTR cleavage fragment in the SOD1G93A model; similar changes are not seen in the Loa mouse. Cocultures of motor neurons and glia expressing mutant SOD1 (mSOD1) in compartmentalized chambers indicate that inhibition of retrograde stress signaling is sufficient to block activation of cellular stress pathways and to rescue motor neurons from mSOD1-induced toxicity. Hence, a shift from survival-promoting to death-promoting retrograde signaling may be key to the rapid onset of neurodegeneration seen in ALS.

Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata)

Transgenic broccoli (Brassica oleracea var. italica) was produced by two Agrobacterium tumefaciens-mediated transformation methods. One used flowering stalk explants from mature plants; the other used hypocotyl and petiole explants from in vitro-grown seedlings. Several hundred transformants containing a Bacillus thuringiensis ∂-endotoxin gene (CryIA(c)-type) and the neomycin phosphotransferase gene were recovered. Rooted transformants were obtained in as little as 3 months using seedling explants. Transgenic cabbage was also obtained by the seedling explant method. Parameters important for high efficiency regeneration and transformation rates included use of a tobacco nurse cell layer, sealing of petri dishes with a porous surgical tape instead of Parafilm, preculture of seedling explants and appropriate length of co-cultivation with Agrobacterium. Advantages and disadvantages of each transformation procedure are discussed.

The Cortical Microtubule Array: From Dynamics to Organization

Cortical microtubules (CMTs) are essential for normal plant morphogenesis because they affect the axes of cell elongation and predict the placement of cell division planes. The function of the CMTs is intimately linked to their organizational state, which is subject to spatial and temporal

Regulation of Dynactin through the Differential Expression of p150Glued Isoforms

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.

Microtubule Severing at Crossover Sites by Katanin Generates Ordered Cortical Microtubule Arrays in Arabidopsis

The noncentrosomal cortical microtubules (CMTs) of land plants form highly ordered parallel arrays that mediate cell morphogenesis by orienting cellulose deposition. Since new CMTs initiate from dispersed cortical sites at random orientations, parallel array organization is hypothesized to require selective pruning of CMTs that are not in the dominant orientation. Severing of CMTs at crossover sites is proposed to be a potential pruning mechanism; however, the parameters and molecular mechanisms underlying this activity are unknown. Here, using live-cell imaging, we show that severing preferentially targets the overlying CMTs at crossover sites and leads to their depolymerization about 85% of the time. In addition, the probability of severing has a sigmoidal relationship to the crossover dwell time, indicating a strong bias for longer-lived crossover sites to be severed. We found that severing at CMT crossover sites was completely abolished in the Arabidopsis katanin mutant. Consistent with this finding, GFP-tagged katanin driven by its native promoter localizes at sites of CMT crossover prior to severing. Furthermore, array recovery experiments showed that CMTs fail to become ordered in the katanin mutant. We conclude that katanin is solely responsible for severing at CMT crossover sites and that this activity is essential to generate ordered CMT arrays.The noncentrosomal cortical microtubules (CMTs) of land plants form highly ordered parallel arrays that mediate cell morphogenesis by orienting cellulose deposition. Since new CMTs initiate from dispersed cortical sites at random orientations, parallel array organization is hypothesized to require selective pruning of CMTs that are not in the dominant orientation. Severing of CMTs at crossover sites is proposed to be a potential pruning mechanism; however, the parameters and molecular mechanisms underlying this activity are unknown. Here, using live-cell imaging, we show that severing preferentially targets the overlying CMTs at crossover sites and leads to their depolymerization about 85% of the time. In addition, the probability of severing has a sigmoidal relationship to the crossover dwell time, indicating a strong bias for longer-lived crossover sites to be severed. We found that severing at CMT crossover sites was completely abolished in the Arabidopsis katanin mutant. Consistent with this finding, GFP-tagged katanin driven by its native promoter localizes at sites of CMT crossover prior to severing. Furthermore, array recovery experiments showed that CMTs fail to become ordered in the katanin mutant. We conclude that katanin is solely responsible for severing at CMT crossover sites and that this activity is essential to generate ordered CMT arrays.

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.