Arp2/3 complex subunit ARPC2 binds to microtubules.

Arp2/3 complex plays a fundamental role in the nucleation of actin filaments (AFs) in yeasts, plants, and animals. In plants, the aberrant shaping and elongation of several types of epidermal cells observed in Arp2/3 complex knockout plant mutants suggest the importance of Arp2/3-mediated actin nucleation for various morphogenetic processes. Here we show that ARPC2, a core Arp2/3 complex subunit, interacts with both actin filaments (AFs) and microtubules (MTs). Plant GFP-ARPC2 expressed in Nicotiana tabacum BY-2 cells, leaf epidermal cells of Nicotiana benthamiana and root epidermal cells of Arabidopsis thaliana decorated MTs. The interaction with MTs was demonstrated by pharmacological approach selectively interfering with either AFs or MTs dynamics as well as by the in vitro co-sedimentation assays. A putative MT-binding domain of tobacco NtARPC2 protein was identified using the co-sedimentation of several truncated NtARPC2 proteins with MTs. Newly identified MT-binding ability of ARPC2 subunit of Arp2/3 complex may represent a new molecular mechanism of AFs and MTs interaction.

Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

Plant cell lines in cell morphogenesis research.

Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation.

Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter.

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes.

Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role.

PIN proteins perform a rate-limiting function in cellular auxin efflux.

Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport-related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux.

Polar PIN localization directs auxin flow in plants.

Polar flow of the phytohormone auxin requires plasma membrane-associated PIN proteins and underlies multiple developmental processes in plants. Here we address the importance of the polarity of subcellular PIN localization for the directionality of auxin transport in Arabidopsis thaliana. Expression of different PINs in the root epidermis revealed the importance of PIN polar positions for directional auxin flow and root gravitropic growth. Interfering with sequence-embedded polarity signals directly demonstrates that PIN polarity is a primary factor in determining the direction of auxin flow in meristematic tissues. This finding provides a crucial piece in the puzzle of how auxin flow can be redirected via rapid changes in PIN polarity.

Methods of dendritic cell preparation for acute lymphoblastic leukemia immunotherapy in children.

Cell immunotherapy through dendritic cells (DC) presents a hopeful strategy for the treatment of various tumors. The aim of our study was to find which progenitor cells are most suitable for the preparation of dendritic cells in acute lymphoblastic leukemia (ALL) in pediatric patients, whether blasts from bone marrow or dendritic cells generated from peripheral blood mononuclear cells taken at the time of remission after induction chemotherapy. DC generated from the BM blasts of patients with B-ALL and T-ALL (n = 15) at the time of diagnosis expressed low levels of costimulatory molecules and CD markers typical for mature DC. In contrast, DC cultivated from peripheral mononuclear cells of patients (n = 9) had comparable morphology and expression of costimulatory molecules to DC obtained from healthy individuals, which was even higher after tumor lysate pulsing. Autologous lymphocyte proliferation increased after DC blasts lysate pulsation and further after lymphocyte restimulation, showing evidence of induction of specific cytotoxic lymphocytes. When comparing both cell sources for the preparation of DC in patients with ALL, it appears that peripheral mononuclear cells obtained after chemotherapy are more suitable than bone marrow leukemic blasts due to similar morphology, phenotypic, and functional capacity to monocytes of healthy donors. Despite this, it is necessary to take into account individual variability when preparing DC-based vaccines. The final verification of the efficiency of immunotherapy against residual hematopoietic malignant cells in patients with ALL can only be obtained through a clinical study.

Local, efflux-dependent auxin gradients as a common module for plant organ formation.

Plants, compared to animals, exhibit an amazing adaptability and plasticity in their development. This is largely dependent on the ability of plants to form new organs, such as lateral roots, leaves, and flowers during postembryonic development. Organ primordia develop from founder cell populations into organs by coordinated cell division and differentiation. Here, we show that organ formation in Arabidopsis involves dynamic gradients of the signaling molecule auxin with maxima at the primordia tips. These gradients are mediated by cellular efflux requiring asymmetrically localized PIN proteins, which represent a functionally redundant network for auxin distribution in both aerial and underground organs. PIN1 polar localization undergoes a dynamic rearrangement, which correlates with establishment of auxin gradients and primordium development. Our results suggest that PIN-dependent, local auxin gradients represent a common module for formation of all plant organs, regardless of their mature morphology or developmental origin.