Arabidopsis VILLIN2 and VILLIN3 act redundantly in sclerenchyma development via bundling of actin filaments

The organization of the actin cytoskeleton has been implicated in sclerenchyma development. However, the molecular mechanisms linking the actin cytoskeleton to this process remain poorly understood. In particular, there have been no studies showing that direct genetic manipulation of the actin cytoskeleton affects sclerenchyma development. Villins belong to the villin/gelsolin/fragmin superfamily and are versatile actin-modifying proteins. Several recent studies have implicated villins in tip growth of single cells, but how villins act in multicellular plant development remains largely unknown. Here, we found that two closely related villin isovariants from Arabidopsis, VLN2 and VLN3, act redundantly in sclerenchyma development. Detailed analysis of cross-sections from inflorescence stems of vln2 vln3 double mutant plants revealed a reduction in stem size and in the number of vascular bundles; however, no defects in synthesis of the secondary cell wall were detected. Surprisingly, the vln2 vln3 double mutation did not affect cell elongation of inter-fascicular fibers. Biochemical analyses showed that recombinant VLN2 was able to cap, sever and bundle actin filaments, similar to VLN3. Consistent with these biochemical activities, loss of function of VLN2 and VLN3 resulted in a decrease in the amount of F-actin and actin bundles in plant cells. Collectively, our findings demonstrate that VLN2 and VLN3 act redundantly in sclerenchyma development via bundling of actin filaments.     

Psychophysiological Mechanisms of Writing Difficulties in Schoolchildren

The temporal and qualitative organization of writing movements was studied in six- to seven-year-old and nine- to ten-year-old right-handed and left-handed children with underdeveloped visuomotor coordination. The characteristic features of the temporal and qualitative organization of these movements at different stages of motor skill formation were found. Regarding the temporal structure of the series of writing movements, a pause was observed between individual movements in the series of motor task formation. The duration of this pause may serve as a criterion of the degree and efficiency of writing skill formation. The disturbance of the temporal structure of movements during the formation of handwriting speed at the initial stages of learning deteriorated the temporal organization of movements, drastically decreased the quality, and impeded the formation of the skill. The destructive processes were more pronounced in the case of accelerated handwriting in left-handed children and especially in children with underdeveloped visuomotor coordination.     

Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.     

Subthalamic lesions eliminate sexual behavior in the male rat

Electrical stimulation of parts of the subthalamus and mesencephalon produces coordinated stepping movements, and for this reason these areas are sometimes referred to as the subthalamic and mesencephalic "locomotor" regions. In this study we contrast the sexual behavioral effect of electrolytic destruction of these two regions in the male rat. Lesions of the mesencephalic locomotor region had no significant effect on male sexual behavior. In contrast, subthalamic lesions centered on the caudal zona incerta just dorsal to the subthalamic nucleus eliminated sexual behavior in 6 of 15 males. The sexual behavior of the remaining males was affected to a lesser degree, for the most part in accord with the extent of destruction to this "critical zone." Subthalamic lesions produced no obvious impairment in locomotion, posture, limb use, muscle tone or sensorimotor orientation. Even so, the fact that electrical stimulation of the subthalamus elicits coordinated stepping suggests that the region is linked with systems directly concerned with movement and locomotion. These links could be particularly important in the process by which sexual motivation is translated into sexual behavior.     

Role of the lateral hypothalamus and prefrontal cortex in organization of voluntary movements

Roles of the lateral hypothalamus and prefrontal cortex in organization and control of automatized food-procuring movements were studied in rats under conditions of unrestrained behavior with the use of video- and photorecording techniques, destruction of the brain structures, and recording of single neuron impulse responses. The lateral hypothalamus is considered a link in the control system for food-procuring movements, which provides formation of the motor program. The role of the prefrontal cortex is regarded as related to programing and initiation of food-procuring movements and current control of efficiency of their performance. Positions of the lateral hypothalamus and prefrontal cortex within an ensemble of the motor centers, which organizes voluntary movements, are discussed.     

The organization of instrumental food-procuring movements in rats]

The roles of the lateral hypothalamus, basolateral nucleus of the amygdalar complex, the second field of the frontal cortex, and ventromedial thalamic nucleus in organization of the fast ballistic food-procuring movements were studied in albino rats. Sequences of uni- and bilateral destruction of the brain structures were assessed by photorecording. Movement-related neuronal activity in these structures was recorded in freely moving animals. A specific involvement of each of the above listed structures in organization of food-procuring movements was shown. The lateral hypothalamus seems to participate in initiation of the motor program and its efficient realization, the basolateral amygdala appears to produce activatory, training, and stabilizing effects. The second field of the motor cortex leads in movement acquisition (i.e., in memorizing) and decision making about triggering the program. The idea about the relay role of the thalamic motor nucleus is supplemented by understanding of its more complex integrative function.     

On the control of stridulation in the acridid grasshopper Omocestus viridulus L.

Summary In tethered, minimally dissected grasshoppers stridulation was elicited by DC brain stimulation. Intracellular recording, stimulation and staining of interneurons in the metathoracic ganglion complex were performed simultaneously with measurements of hindleg movements. The functional significance of interneurons for generation of the stridulation rhythm and bilateral coordination of the hindlegs was tested and quantitatively analysed. Interneurons involved in stridulation were found in the metathoracic and abdominal neuromeres. Typically they included arborizations in a dorsal neuropil parallel to the long axis of the ganglion. Interneuron T3-LI-3 started and drove the ipsilateral rhythm generator. The stridulation rhythm was reset by interneuron A1-AC-1. It was stopped if interneuron T3-AC-1 was stimulated. Bilateral coordination of stridulation movements was based on excitatory and inhibitory pathways between the hemiganglionic networks. Switching in the coordination of hindleg movement patterns was induced by changes in the discharge rate of the bilateral arborizing interneuron T3-LC-4. The hemiganglionic interneurons T3-LI-3 and T3-LI-1 influenced coordination via the activity level within the networks.     

The psychomotor test for research of eye-hand coordination at performance of monotonous activity on tracking target

Visual-motor coordination is necessary for successful performance of everyday activities. Many tasks, such as driving or operating devices in the workplace, require a variety of coordination patterns with different levels of compatibility between the eyes and the hand. The psychomotor test was developed which makes it possible to analyze visual-motor coordination disorders caused by a decrease in the level of wakefulness. A small circular target (14 mm in diameter) was moving with a low constant velosity (12 mm/s) in a circular trajectory (80 mm in diameter) with a period of 20 s. Subjects were instructed to keep the mouse-driven cursor inside a target, overstepping the limits of the moving target was considered as an error. To test the attention level, an additional stimulus was introduced which appeared for 3 seconds with an interval of 15 to 40 s. When the stimulus appeared, it was required to touch it with the cursor and click the mouse button. Cursor trajectories have a temporal resolution of 120 Hz. Eye movements were recorded with a PC-based Eyegaze Development System (LC Technologies, USA) measuring corneal reflectance with a collection rate of 120 Hz. Monotonous character of the test performance induced drowsiness and led to errors 25-30 minutes after the beginning of the experiment. Changes in physiological vigilance level was evaluated with EEG recording. Analysis of the dynamic characteristics of smooth and saccadic eye movements and hand movements showed their high sensitivity to a decrease in efficiency of operator's activity caused by a drop in the level of wakefulness. It is suggested that further development of this approach to measuring eye-hand coordination will promote working out a contactless method for the express diagnostics of the critical levels of drowsiness as well as for the definition of professional characteristics of an operator.     

The role of tensile fields and contact cell polarization in the morphogenesis of amphibian axial rudiments

Summary The role of stretching-generated tensile stresses upon the organization of axial rudiments have been studied. Pieces of the dorsal wall ofXenopus laevis andRana temporaria embryos at the late gastrula stage were rotated through 90°, transplanted into the field of neurulation tensions of another embryo and replaced by ventral tissues already insensitive to inductive influences. The axial rudiments which developed from rotated and transplanted dorsal tissues oped from rotated and transplanted dorsal tissues almost completely reorientated according to the tensile patterns in adjacent host tissues. Some of the donor cells changed their presumptive fates in accordance with their new positions in the host tensile field. Transplanted ventral tissues were involved in the morphogenetic movements specific for the dorsal regions and imitated some typical dorsal structures. In the regions without pronounced tensions the structure of transplanted axial rudiments was chaotic. It is suggested that the organization of the axial structures is established and maintained by tensile fields created by uniformly polarized cells. Cell polarization can be transmitted by contact from host to donor tissues. The specificity of this propagating process and its morphogenetical role is discussed.     

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). "Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus." Development. 126, 3703-3713.]. We present a revised model of gastrulation movements in Xenopus laevis.     

Calcium signaling during convergent extension in Xenopus

BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.     

Arabidopsis VILLIN1 and VILLIN3 Have Overlapping and Distinct Activities in Actin Bundle Formation and Turnover

Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca²⁺ insensitive. Based on phylogenetic analyses and conservation of Ca²⁺ binding sites, we predict that VLN3 is a Ca²⁺-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities.     

Blockade of a motor response by cholinergic stimulation of the toad midbrain tegmentum

Application of an acid solution to the dorsal skin of conscious toads having intact nervous system induces a scratching reflex and escape movements, as well as autonomic alterations (hypertension and tachycardia) that are part of the defense response. The motor components of this response are abolished or reduced by microinjection of 60, 30, 15 or 7.5 ng carbachol into the midbrain tegmentum. The cardiovascular components, however, continue to be present, although their amplitude is reduced. The depression of the motor response is statistically significant up to 15 minutes for the 60 ng dose, up to 10 minutes for the 15 and 30 ng doses, and only up to 5 minutes for the 7.5 ng dose. The data suggest that the midbrain tegmentum may modulate the reflex motor response triggered by a noxious stimulus and also participate in the organization of the escape movements. The importance of cholinergic agents in this modulation is discussed. The persistence of the cardiovascular component of the response shows the importance of this parameter as an indicator of alert situations.     

Visual position stabilization in the hummingbird hawk moth, Macroglossum stellatarum L. I. Behavioural analysis

Optomotor responses of freely flying hawk moths, Macroglossum stellatarum, were characterized while the animals were hovering in front of and feeding on a dummy flower. Compensatory translational and rotational movements of the hawk moth were elicited by vertical grating patterns moving horizontally, mimicking imposed rotational and translational displacements of the animal in the horizontal plane. Oscillatory translational and rotational pattern motion leads to compensatory responses that peak in the frequency range between 2 Hz and 4 Hz. The control systems mediating the translational and rotational components of the optomotor response do not seem to influence each other. The system mediating translational responses is more sensitive in the fronto-lateral part of the visual field than in the lateral part; the opposite is true for the rotational system. The sensitivity of the translational system does not change along the vertical, whereas the rotational system is much more sensitive to motion in the dorsal than in the ventral part of the visual field. These sensitivity gradients may reflect an adaptation to the specific requirements of position stabilization in front of flowers during feeding. Accepted: 13 August 1997     

Organization of a program of automatized food-procuring movement in rats

With the use of techniques of video- and photorecording, local destruction of the brain structures, and recording of the spike responses, roles of the suprasegmental brain structures in organization and control of automatized food-procuring movements were studied in rats under conditions of unrestrained behavior. It is shown that there are several neuronal populations in the prefrontal cortex related to programing, initiation, and current control of the efficacy of performance of food-procuring movements. The lateral hypothalamus is considered a motivational structure. It acts as the source providing an increase in the excitability and further support of the excitation level in the structures, which are links of the systems controlling food-procuring movements. The lateral hypothalamus is also involved in a system providing formation and final application of motor programs.     

Activation of the Shoulder-Belt and Shoulder Muscles in Two-Joint Arm Movements Performed in Humans with the Action of Opposite Loadings

In tests on four volunteers, we examined coordination of central motor commands (CMCs) controlling slow two-joint movements of the arm within the horizontal plane. Current amplitudes of EMGs recorded from six muscles of the shoulder belt and shoulder and subjected to full-wave rectifying and low-frequency filtration were considered correlates of these commands. In particular, we studied the dependence of coordination of CMCs on the direction of an external force applied to the distal forearm part. As was found, coordination of CMCs significantly depends on the direction of the force flexing the elbow joint. According to our observations, EMGs of definite muscles in the case of performance of a two-joint movement can, in a first approximation, be presented as linear combinations of the EMGs recorded in the course of separate sequential single-joint movements under conditions of shifting the reference point of the hand toward the same point of the operational space as that in the two-joint movement. These data can be interpreted as confirmation of the principle of superposition of elementary CMCs in the performance of complex movements of the extremity.     

Disruption of food-getting movements after removal of the motor or premotor zone of the cerebral cortex in cats]

Instrumental food-procuring movements were studied in cats before and after unilateral or bilateral ablation of the motor or premotor cortical area. It is shown that unilateral impairment of the motor area affects the strength and accuracy of movements of the contralateral fore-leg, whereas the ablation of the premotor area leads to a slowing down of movements and breaking of a goal-directed movement into separate components. Bilataral ablation of the motor area irreversibly abolished the instrumental reflex. The ablation of the premotor cortex destroyed the animal's reaction to the sound signal, but food-procuring movements of the fore-legs were disturbed only temporarily. The obtained data are discussed on the basis of the concept that in cats the above cortical areas play different roles in the organization of goal directed behaviour.     

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originatefrom the axis of organelles and cytoskeletal elements establishedin the oocyte as it divides from the oogonium. Along this axis,cytoplasmic materials are localized during oogenesis: yolk platelets,for example, are translocated toward the vegetal pole, increasingtheir amount and size in that region. In the first cell cycleafter fertilization, the egg cortex rotates 30° relativeto the cytoplasmic core, modifying animal-vegetal organization.The direction of this rotation, biased by the point of spermentry, defines the site of development of anatomical structuresof the dorsal midline of the embryo. As its immediate effect,rotation activates the cytoplasm of a subregion of the vegetalhemisphere, causing cells cleaved from this subregion to bemore effective than other vegetal parts in inducing marginalzone cells to initiate gastrulation movements. The most stronglyinduced part of the marginal zone begins gastrulation first(the dorsal lip of the blastopore) and proceeds through a seriesof cell interactions leading to its determination as the anteriordorsal mesoderm of the embryo. If these cell movements are inhibitedin the gastrula stage, or if vegetal induction is inhibitedin the blastula stage, or if cortical rotation is inhibitedin the first cell cycle after fertilization, the embryo alwaysfails to develop dorsal structures of the anterior end of itsbody axis; the more inhibition, the more posterior is the levelof truncation, until a radial ventralized embryo develops, derivedfrom the animal-vegetal organization of the oocyte.