Insects Use Two Distinct Classes of Steps during Unrestrained Locomotion

Background

Adaptive, context-dependent control of locomotion requires modulation of centrally generated rhythmic motor patterns through peripheral control loops and postural reflexes. Thus assuming that the modulation of rhythmic motor patterns accounts for much of the behavioural variability observed in legged locomotion, investigating behavioural variability is a key to the understanding of context-dependent control mechanisms in locomotion. To date, the variability of unrestrained locomotion is poorly understood, and virtually nothing is known about the features that characterise the natural statistics of legged locomotion. In this study, we quantify the natural variability of hexapedal walking and climbing in insects, drawing from a database of several thousand steps recorded over two hours of walking time.

Results

We show that the range of step length used by unrestrained climbing stick insects is large, showing that step length can be changed substantially for adaptive locomotion. Step length distributions were always bimodal, irrespective of leg type and walking condition, suggesting the presence of two distinct classes of steps: short and long steps. Probability density of step length was well-described by a gamma distribution for short steps, and a logistic distribution for long steps. Major coefficients of these distributions remained largely unaffected by walking conditions. Short and long steps differed concerning their spatial occurrence on the walking substrate, their timing within the step sequence, and their prevalent swing direction. Finally, ablation of structures that serve to improve foothold increased the ratio of short to long steps, indicating a corrective function of short steps.

Conclusions

Statistical and functional differences suggest that short and long steps are physiologically distinct classes of leg movements that likely reflect distinct control mechanisms at work.     

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a₂ position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca₁a₂X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a₂ and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.     

Two Maize Genes Are Each Targeted Predominantly by Distinct Classes of Mu Elements

The Mutator transposable element system of maize has been used to isolate mutations at many different genes. Six different classes of Mu transposable elements have been identified. An important question is whether particular classes of Mu elements insert into different genes at equivalent frequencies. To begin to address this question, we used a small number of closely related Mutator plants to generate multiple independent mutations at two different genes. The overall mutation frequency was similar for the two genes. We then determined what types of Mu elements inserted into the genes. We found that each of the genes was preferentially targeted by a different class of Mu element, even when the two genes were mutated in the same plant. Possible explanations for these findings are discussed. These results have important implications for cloning Mu-tagged genes as other genes may also be resistant or susceptible to the insertion of particular classes of Mu elements.     

Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients.     

Tubulin Stability and Decay: Mediation by Two Distinct Classes of IKP104-Binding Sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both and subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the and subunits of tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (_ss). Moreover, IKP104 lowers the sulfhydryl titer of _ss tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of _ss tubulin. The cleavage at the C-terminal ends of both and modulates the effect of IKP104 on the subunit, but not on the subunit. Fluorometric binding data analysis suggests that IKP104 binds to the _ss tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for and and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of and and the effect of the high-affinity site is restricted largely to the subunit, while the low-affinity-site binding is modulated by the C-terminal domain of . It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.     

Anticancer Properties of Distinct Antimalarial Drug Classes

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC₅₀s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC₅₀s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.     

Soluble Amyloid Precursor Protein Alpha Interacts with alpha3-Na,K-ATPAse to Induce Axonal Outgrowth but Not Neuroprotection: Evidence for Distinct Mechanisms Underlying these Properties

Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble N-terminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPα-induced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.     

A Conceptual Mathematical Model of the Dynamic Self-Organisation of Distinct Cellular Organelles

Formation, degradation and renewal of cellular organelles is a dynamic process based on permanent budding, fusion and inter-organelle traffic of vesicles. These processes include many regulatory proteins such as SNAREs, Rabs and coats. Given this complex machinery, a controversially debated issue is the definition of a minimal set of generic mechanisms necessary to enable the self-organization of organelles differing in number, size and chemical composition. We present a conceptual mathematical model of dynamic organelle formation based on interacting vesicles which carry different types of fusogenic proteins (FP) playing the role of characteristic marker proteins. Our simulations (ODEs) show that a de novo formation of non-identical organelles, each accumulating a different type of FP, requires a certain degree of disproportionation of FPs during budding. More importantly however, the fusion kinetics must indispensably exhibit positive cooperativity among these FPs, particularly for the formation of larger organelles. We compared different types of cooperativity: sequential alignment of corresponding FPs on opposite vesicle/organelles during fusion and pre-formation of FP-aggregates (equivalent, e.g., to SNARE clusters) prior to fusion described by Hill kinetics. This showed that the average organelle size in the system is much more sensitive to the disproportionation strength of FPs during budding if the vesicular transport system gets along with a fusion mechanism based on sequential alignments of FPs. Therefore, pre-formation of FP aggregates within the membranes prior to fusion introduce robustness with respect to organelle size. Our findings provide a plausible explanation for the evolution of a relatively large number of molecules to confer specificity on the fusion machinery compared to the relatively small number involved in the budding process. Moreover, we could speculate that a specific cooperativity which may be described by Hill kinetics (aggregates or Rab/SNARE complex formation) is suitable if maturation/identity switching of organelles play a role (bistability).     

Axonal Transport of Zinc Transporter 3 and Zinc Containing Organelles in the Rodent Adrenergic System

Zinc is the second most abundant trace metal (after iron) in mammalian tissues, and it is an essential element for growth, development, DNA synthesis, immunity, and other important cellular processes. A considerable amount of zinc in the brain exists as a pool of free or loosely bound zinc ions in synaptic vesicles with zinc transporter 3 (ZnT3) in their membranes. Here we demonstrate that also in the peripheral sympathetic nervous system zinc handling neurons exist. In autonomic ganglia of rats and mice a subset of neuronal cell bodies contain zinc, visualized by the autometallographic (AMG) and TSQ histochemical methods. The Zn-transporter 3 is, as shown by immunofluorescence, also present in tyrosine hydroxylase (TH)-positive neurons, but rarely in cell bodies with neuropeptide Y (NPY)-immunoreactivity (IR). In axons of crush-operated sciatic nerves a rapid bidirectional accumulation of AMG granules occurred. Also ZnT3-IR was found to accumulate rapidly in anterograde as well as retrograde direction, colocalized with TH-IR. So far nerve terminals with ZnT3-IR have not been observed. The functional significance of zinc ions in the sympathetic system is not known. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.     

Molecular Modeling Indicates that Two Chemically Distinct Classes of Anti-Mitotic Herbicide Bind to the Same Receptor Site(s)

Dinitroaniline and phosphorothioamidate herbicides disrupt microtubule assembly from tubulin protein dimers and thereby halt microtubule-based processes such as mitosis in plant cells. Despite the contrasting chemical properties of dinitroaniline and phosphorothioamidate herbicides, a three-dimensional molecular analysis revealed remarkable electrostatic similarity between these two classes of herbicide. From these data it is proposed that dinitroaniline and phosphorothioamidate herbicides share common binding site(s) in the plant cell.     

Differences in the Kinetics of Axonal Transport for Individual Lipid Classes in Rat Sciatic Nerve

Lipid precursors ([2-3H]glycerol for phospholipids and [3H]acetate for cholesterol) were injected into the L-5 dorsal root ganglion of adult rats. At various times, animals were killed, the ganglion and consecutive 5-mm segments of sciatic nerve were dissected, and lipids were extracted and analyzed by TLC. Individual lipid classes exhibited markedly different transport patterns. The crest of radioactive phosphatidylcholine moved as a sharply defined front at about 300 mm/day, with a relatively flat plateau behind the moving crest. Although some radioactive phosphatidylethanolamine also moved at the same rate, the crest was continually attenuated as it moved so that a gradient of radioactive phosphatidylethanolamine along the axon was maintained for several days. Transported diphosphatidylglycerol exhibited a defined crest, as did phosphatidylcholine, but moved at about half the rate. Labeled cholesterol was transported at a rapid rate similar to that for phosphatidylcholine and phosphatidylethanolamine, but like phosphatidylethanolamine, the initial moving crest of radioactivity was continually attenuated. Relative to the phospholipids, cholesterol showed a more prolonged period of accumulation in the axons and was more metabolically stable. We propose that most labeled phosphatidylcholine, phosphatidylethanolamine, and cholesterol is transported in similar (or the same) rapidly moving membranous particles. Once incorporated into these particles, molecules of phosphatidylcholine tend to maintain associated with them during transport. In contrast, molecules of phosphatidylethanolamine and cholesterol in these transported particles exchange extensively with unlabeled molecules in stationary axonal structures. Diphosphatidylglycerol, localized in a specialized organelle, the mitochondrion, is transported at a slower rate than other phospholipids, and does not exchange with other structures.