A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

Hyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 µm away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport.Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein ‘upregulated during mitosis’, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

Microtubule associated protein WAVE DAMPENED2-LIKE (WDL) controls microtubule bundling and the stability of the site of tip-growth in Marchantia polymorpha rhizoids

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes–WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)–are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability.

Modeling of growth in shoot apical dome

The vectorial field of displacement velocities V in growing apical dome is calculated from the scalar field adjusted to the geometry of the dome and to different variants of the distribution of linear growth rate along axis. The distribution of growth rate in volume and the temporal course of cell wall net deformation in the apical dome calculated from the V fit within the range of empirical data when the linear growth rate along the axis increases with distance from the tip. The distribution of volume growth rate attains the minimum inside the distal region of the dome where the zone of central mother cells occurs.

Two algorithms of determining the middle point of the shoot apex by surrounding organ primordia positions and their usage for computer measurements of divergence angles

The measurements of the divergence angle between organ primordia in magnolia floral apices and vegetative apices (embryonic shoots) of coniferous trees were performed, using computer application Phyl for IRIX. It appears that the values of the angle are strongly affected by the position of the apex centre, which was calculated by the program on a base of input positions of surrounding primordia. Two algorithms were used to determine the centre position: in program version Phyl 1 it was calculated as the gravity centre whereas in Phyl 2 version as a geometrical midpoint. The both methods differ in resulting centre position. This is dependent on such apex features as: proportions between its size and the size of primordia (expression of phyllotaxis), the angular distances between succesive primordia (quality of phyllotaxis), radial distances between primordia of different age (profile of the apical dome), the number of recorded primordia. The accuracy and reliability of both algorithms for determination of the centre position and, consequently, the reliability of divergence measurements were verified by applying them to ideal, computer simulated spiral phyllotactic systems with divergence given arbitrarily by the user. The conclusions from the results of simulations are that there is a possibility of selecting more suitable algorithm for particular apex with regard to specific parameters of its phyllotactic system.

Modeling of spatial variations of growth within apical domes by means of the growth tensor. L Growth specified on dome axis

By using the growth tensor and a natural curvilinear coordinate system for description of the distribution of growth in plant organs, three geometric types of shoot apical domes (parabolic, elliptical and hyperbolic) were modeled. It was assumed that apical dome geometry remains unchanged during growth and that the natural coordinate systems are paraboloidal and prolate spheroidal. Two variants of the displacement velocity fields V were considered. One variant is specified by a constant relative elemental rate of growth along the axis of the dome. The second is specified by a rate increasing proportionally with distance from the geometric focus of the coordinate systems (and the apical dome). The growth tensor was used to calculate spatial variations of growth rates for each variant of each dome type. There is in both variants a clear tendency toward lower growth rates in the distal region of the dome. A basic condition for the existence of a tunica is met.

Growth and development of shoot apex in barley II. Distribution of growth rates during vegetative phase

The changes in the volume of the apical dome and of the frusta indicate that the mean relative rate of volume growth of the whole apical dome is much higher than in the first frustum which, in turn, grows much faster than the next one. It was found in studies of the distribution of mitoses and of cell arrangement that the volume relative growth rate of the distal part of the apical dome is at least 5-10 times slower than that at the level of new leaf primordia initiation.

Ontogenetic variation of phyllotaxis and apex geometry in vegetative shoots of Sedum maximum (L.) Hoffm.

Phyllotaxis of vegetative shoots of <em>Sedum maximum</em> (L.) Hoffm. is variable and unstable. Phyllotactic transitions proceeding either between two whorled, two spiral or a whorled and a spiral pattern occur during shoot ontogeny. A general rule is that in the course of these transitions numbers of contact parastichies increase or much less often diminish. During most common transitions only a single parastichy is added to or subtracted from the already existing contact parastichy pattern. This applies both to the transitions between a spiral and a whorled and to those between two spiral patterns. More parastichies appear only when phyllotaxis changes from one whorled pattern to another. These transitions, however, are the least common in <em>S. maximum</em>. Parameters of apex geometry differ significantly in shoots exhibiting different contact parastichy numbers. The sequence of phyllotactic patterns arranged according to an increasing apical dome area or area ratio, as well as decreasing plastochron ratio (both as defined by Richards) is the same as their sequence based on increasing numbers of contact parastichies. This in turn is the sequence of patterns as they appear during shoot ontogeny. The only parameter which remains relatively constant is the area of the youngest leaf primordium. This implies that during phyllotactic transitions in <em>S. maximum</em> the area ratio changes mainly due to the increase of the apical dome. It seems that in <em>S. maximum</em> this ontogenetic increase of the apical dome, which on the other hand is typical for many plants, somehow differs from these cases where such a change alters only contact parastichy numbers within the same phyllotactic series.

Geometry of shoot apical dome and distribution of growth rates

The distribution of the relative elementary rate of growth (RERG) in apical domes of various shapes and patterns of displacement lines can be analytically examined. The geometry of these domes may be described by parabolas of <em>n</em>-th order, the variant of the distribution of linear growth rate should be established along any displacement line (e.g. along the axis) and then the RERG can be studied as the function depending on the position coordinates and the parameter n. Such investigations of several aplical domes of various shapes have been performed. The results confirm the occurrence of the minimum of relative, elementary growth rate (in volume) in the subapical region of the dome independently of the type of geometry (<em>n</em> parabola order).

Early increased expression of a cyclin-dependant protein kinase (LtCDKA1;1) during inflorescence initiation of the long day grass Lolium temulentum

Knowing where and when different genes express at the shoot apex during the transition to flowering will help in understanding this developmental switch. The CDKA family of serine/threonine kinase genes are appropriate candidates for such developmental switching as they are involved in the regulation of the G1/S and G2/M boundaries of the cell cycle (see review by Dudits et al. 2007) and so could regulate increases of cell division associated with flowering. Furthermore, in rice stems the gibberellin (GA) class of plant growth regulators rapidly upregulate CDKA expression and cell division. Thus, CDKA expression might be linked to the florigenic action of GA as a photoperiodically-generated, signal. For the grass Lolium temulentum L., we have isolated an LtCDKA1;1 gene, which is upregulated in shoot apices collected soon after the start of a single florally inductive long day (LD). In contrast to weak expression of LtCDKA1;1 in the vegetative shoot apex, in situ and PCR-based mRNA assays and immunological studies of its protein show very rapid increases in the apical dome at the time that florigenic signals arrive at the apex (<6 h after the end of the LD). By ~54 h LtCDKA1;1 mRNA is localised to the floral target cells, the spikelet primordia. Later both LtCDKA1;1 mRNA and protein are most evident in floret meristems. Only ~10% of cells within the apical dome are dividing at any time but the LD increase in LtCDKA1;1 may reflect an early transient increase in the mitotic index (Jacqmard et al. 1993) as well as a later increase when spikelet primordia form. Increased expression of an AP1-like gene (LtMADS2) follows that of LtCDKA1;1. Overall, LtCDKA1;1 is a useful marker of both early florigenic signalling and of later morphological/developmental aspects of the floral transition.