The Interplay of Structural and Cellular Biophysics Controls Clustering of Multivalent Molecules

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. It is not well understood, however, how molecular structure and cellular organization control clustering. Using coarse-grained kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1, and the actin nucleation promoting factor NWASP. Steady-state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching) but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.     

Independent and combined abiotic stresses affect the physiology and expression patterns of DREB genes differently in stress‐susceptible and resistant genotypes of banana

In tropics, combined stresses of drought and heat often reduce crop productivity in plants like Musa acuminata L. We compared responses of two contrasting banana genotypes, namely the drought‐sensitive Grand Nain (GN; AAA genome) and drought tolerant Hill banana (HB; AAB genome) to individual drought, heat and their combination under controlled and field conditions. Drought and combined drought and heat treatments caused greater reduction in leaf relative water content and greater increase in ion leakage and H₂O₂ content in GN plants, especially in early stages, while the responses were more pronounced in HB at later stages. A combination of drought and heat increased the severity of responses. Real‐time expression patterns of the A‐1 and A‐2 group DEHYDRATION‐RESPONSIVE ELEMENT BINDING (DREB) genes revealed greater changes in expression in leaves of HB plants for both the individual stresses under controlled conditions compared to GN plants. A combination of heat and drought, however, activated most DREB genes in GN but surprisingly suppressed their expression in HB in controlled and field conditions. Its response seems correlated to a better stomatal control over transpiration in HB and a DREB‐independent pathway for the more severe combined stresses unlike in GN. Most of the DREB genes had abscisic acid (ABA)‐responsive elements in their promoters and were also activated by ABA suggesting at least partial dependence on ABA. This study provides valuable information on physiological and molecular responses of the two genotypes to individual and combined drought and heat stresses.     

Association between risk of oral precancer and genetic variations in microRNA and related processing genes

Background

MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease.

Results

Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3–0.9) and OR = 0.7(0.5–0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3–1) and OR = 0.68(0.5–0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1–3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database.

Conclusion

Variations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.     

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (μ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by μ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.     

The EAR Motif Controls the Early Flowering and Senescence Phenotype Mediated by Over-Expression of SlERF36 and Is Partly Responsible for Changes in Stomatal Density and Photosynthesis

The EAR motif is a small seven amino acid motif associated with active repression of several target genes. We had previously identified SlERF36 as an EAR motif containing gene from tomato and shown that its over-expression results in early flowering and senescence and a 25–35% reduction of stomatal density, photosynthesis and stomatal conductance in transgenic tobacco. In order to understand the role of the EAR motif in governing the phenotypes, we have expressed the full-length SlERF36 and a truncated form, lacking the EAR motif under the CaMV35S promoter, in transgenic Arabidopsis. Plants over-expressing the full-length SlERF36 show prominent early flowering under long day as well as short day conditions. The early flowering leads to an earlier onset of senescence in these transgenic plants which in turn reduces vegetative growth, affecting rosette, flower and silique sizes. Stomatal number is reduced by 38–39% while photosynthesis and stomatal conductance decrease by about 30–40%. Transgenic plants over-expressing the truncated version of SlERF36 (lacking the C-terminal EAR motif), show phenotypes largely matching the control with normal flowering and senescence indicating that the early flowering and senescence is governed by the EAR motif. On the other hand, photosynthetic rates and stomatal number were also reduced in plants expressing SlERF36ΔEAR although to a lesser degree compared to the full- length version indicating that these are partly controlled by the EAR motif. These studies show that the major phenotypic changes in plant growth caused by over-expression of SlERF36 are actually mediated by the EAR motif.     

Maximizing binding capacity for protein A chromatography

Advances in cell culture expression levels in the last two decades have resulted in monoclonal antibody titers of ≥10 g/L to be purified downstream. A high capacity capture step is crucial to prevent purification from being the bottleneck in the manufacturing process. Despite its high cost and other disadvantages, Protein A chromatography still remains the optimal choice for antibody capture due to the excellent selectivity provided by this step. A dual flow loading strategy was used in conjunction with a new generation high capacity Protein A resin to maximize binding capacity without significantly increasing processing time. Optimum conditions were established using a simple empirical Design of Experiment (DOE) based model and verified with a wide panel of antibodies. Dynamic binding capacities of >65 g/L could be achieved under these new conditions, significantly higher by more than one and half times the values that have been typically achieved with Protein A in the past. Furthermore, comparable process performance and product quality was demonstrated for the Protein A step at the increased loading. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1335–1340, 2014     

Chlorophyllase in <Emphasis Type="Italic">Piper betle</Emphasis> L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown

Total chlorophyll content and chlorophyllase (chlorophyll-chlorophyllido hydrolase EC 3.1.1.14) activity in fresh leaves of Piper betle L. landrace KS was, respectively, twofold higher and eight fold lower than KV, showing negative correlation between chlorophyll and chlorophyllase activity. Specific chlorophyllase activity was nearly eightfold more in KV than KS. ORF of 918 nt was found in cloned putative chlorophyllase cDNAs from KV and KS. The gene was present as single copy in both the landraces. The encoded polypeptide of 306 amino acids differed only at two positions between the KV and KS; 203 (cysteine to tyrosine) and 301 (glutamine to glycine). Difference in chlorophyllase gene expression between KV and KS was evident in fresh and excised leaves. Up regulation of chlorophyllase gene by ABA and down regulation by BAP was observed in both the landraces; however, there was quantitative difference between KV and KS. Data suggests that chlorophyllase in P. betle is involved in chlorophyll homeostasis and chlorophyll loss during post harvest senescence.     

The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left

Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.The founding member of the kinesin superfamily, the cargo-transporting kinesin-1, has been studied in great detail. Dimeric kinesin-1 constructs 1) are mechanical processive, taking ∼100 of 8-nm steps in a hand-over-hand fashion without detaching from the microtubule; and 2), walk parallel to the axis of microtubule protofilaments as they step from one tubulin dimer to the next. The latter was inferred from gliding motility assays, where microtubules propelled by motors bound to a planar substrate surface rotated around their longitudinal axis with periodicities corresponding to the helical course of the protofilaments in supertwisted microtubules (1,2). Interestingly, protofilament tracking of kinesin-1 is lost in nonprocessive, monomeric constructs (3). There, and also for other nonprocessive microtubule motors such as the kinesin-14 Ncd (4) or axonemal dynein (5), significantly shorter pitches of microtubule rotations in gliding motility assays were observed. As suggested previously (6) this may indicate that protofilament tracking is an inherent property of processive microtubule motors.To explore this idea further, we investigated the rotations of 14-protofilament microtubules (left-handed helical pitch of ∼8 μm (2)) in gliding motility assays using kinesin-8 that has been observed to perform ≈12 μm-long processive runs in vitro (7). Streptavidin-coated quantum dots (QDs), sparsely bound to the microtubules, served as reporters of microtubule rotations (Fig. 1 A). Information on the three-dimensional paths of the QDs—and thus on microtubule rotations—were obtained from 1), two-dimensional tracking of the QDs with nanometer precision in x and y (8), in combination with 2), z information derived from fluorescence-interference contrast (FLIC) (2) (Fig. 1, B–D). FLIC originates from destructive and constructive interference effects close to reflecting surfaces and gives rise to a modulation of the detected intensity of a fluorescent object depending on its height above the surface. Specifically, the microtubule-attached QDs appear dark when they are in close proximity to the surface (i.e., when being located between the microtubule and the surface) but brighten up significantly when being further away (i.e., when on the microtubule lattice pointing away from the surface). In our experiments, we observed counterclockwise rotations (looking from the trailing microtubule plus-end in the direction toward the leading minus-end) with an average pitch of 0.93 μm ± 0.20 μm (mean ± SD, N = 75; N is the number of complete rotations obtained from 15 gliding microtubules). Considering the geometry of the assay, the counterclockwise directionality of the rotations corresponds to the motors stepping with a perpetual bias (∼1 protofilament switch event per forward movement over 10 tubulin dimers) toward the left.Open in a separate windowFigure 1Monitoring Kip3-driven microtubule rotations in gliding motility assays. (A) Schematic of the experimental setup. Imaging is performed on top of a reflective silicon surface using fluorescence interference contrast (FLIC) microscopy (2). (B) Maximum projection of the fluorescence signal of a microtubule-attached quantum dot in the Kip3 gliding motility assay. (C) FLIC intensity (red) and lateral distance from the microtubule path (blue) of the quantum dot shown in panel B versus traveled distance along the microtubule path. The periodic FLIC signal is indicative of repeated up- and down-motion. (D) Schematic of the deduced Kip3 path (red) in comparison to the protofilament axis (green) on a 14-protofilament microtubule.The behavior observed in our experiments is in stark contrast to kinesin-1, for which counterclockwise rotations with an average pitch of 7.9 μm were previously observed using the same experimental technique (2). Consequently, the question arises: which structural determinants decide whether a kinesin acts as a strict protofilament tracker (and—if it does not—from where the directional bias of the off-axis stepping originates)? Assuming motility in a hand-over-hand fashion, it will matter which binding sites on the microtubule lattice are within reach of the forward swinging motor head. This reach is primarily set by the neck linkers, the structural elements that connect the two motor heads to the coiled-coil neck domain. More precisely, the reach is a function of the length of the neck linkers and their three-dimensional path dictated by the volumes that are occupied by the motor heads when bound to the microtubule. Based on primary sequence alignment between Kip3 with other members of the kinesin-8 family and prediction of the start of the coiled-coil dimerization domain with the program PCOILS, we assigned the neck linker region to the amino acids K436–H452 (i.e., 17 amino acids). Accounting for neck linker docking of the rear motor head (K436–Q447) (9), the corresponding length of the neck linkers between both heads, composed of five amino acids from the undocked part of the rear-head neck linker and 17 amino acids from the front-head neck linker, is estimated to be 85 Å (see the Supporting Material).We then modeled all configurations of Kip3 with both heads bound simultaneously to adjacent tubulin dimers (Fig. 2, A and B, and see the Supporting Material). The estimated three-dimensional distances between the positions where the neck linkers protrude from the motor heads, respecting the volumes of the heads (Fig. 2 C, gray column; see also the Supporting Material), are measures for the minimally required neck linker lengths for each two-head bound configuration. Comparison between the three-dimensional distances obtained from the model and the available neck linker length (85 Å) suggests that a forward-swinging Kip3 head can most readily reach the tubulin dimer in the front (53 Å needed) and can switch to the protofilament on the left (79 Å for left and 93 Å for front-left needed), but it has difficulties in stepping to the protofilament on the right, which would require a longer neck-linker than it actually exhibits (103 Å for front-right and 105 Å for right needed). The main reason why these long neck-linker distances are required (i.e., >100 Å) is that, to reach the tubulin dimer on the right (or front-right), the neck linker has to bend over the humpy back of the front head (see Fig. 2 B, right and front-right). On the contrary, to reach the tubulin dimer on the left (or front-left), this detour is avoided (see Fig. 2 B, left and front-left). The model-derived preference for left-stepping over right-stepping is in agreement with our experimental observations.Open in a separate windowFigure 2Virtual three-dimensional reconstruction of Kip3 stepping. (A) Tubulin dimer: composed of alpha-tubulin (α) and beta-tubulin (β) monomers, with the unstructured surface-exposed E-Hooks (e). Kip3 front head: Shown with undocked neck linker (U) and following coiled-coil (cc) dimerization domain. Kip3 rear head: Shown with docked (D) and undocked (U) neck linker parts. (B) Illustration of different Kip3 configurations bound with both heads to adjacent tubulin dimers (first heptad repeat of the coiled coil region is artificially unfolded to illustrate all binding configurations). (C) Estimated three-dimensional distances between the positions where the neck linkers protrude from the motor heads, respecting the volumes of the heads (nomenclature as in panel B). For comparison, the direct distances between the tubulin dimers are given.Modeling as described can be applied for kinesin-1, whose neck linkers are three-amino-acids shorter than the neck linkers of Kip3. Whereas the modeled minimally required neck linker lengths for each two-head-bound configuration are almost identical to the values for Kip3, we estimate an available neck linker length of 63 Å (see the Supporting Material), which explains the strict forward stepping of kinesin-1.In summary, we have shown what to our knowledge is the first example of a highly processive kinesin motor (run length of several μm) switching between protofilaments of microtubules. Our modeling suggests that protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1 so that it is able to reach the extra distance required to change protofilaments. The leftward bias cannot be explained by the geometry of the microtubule lattice alone (Fig. 2 C, last column) but follows from the additional consideration of the asymmetric geometry of the motor neck linker complex. A leftward torque component, which may be present in the powerstrokes of the individual heads (3,4), may further promote the leftward bias but is not strictly necessary. While our results were under review, left-handed spiraling along microtubules of beads coated with a modified kinesin-1 (with extended neck linkers (10)) was reported (11); the handedness of the bead rotations is consistent with the handedness of our microtubule rotations and our model.Our results may also provide an alternative explanation for the short-pitch, counterclockwise rotations of microtubules gliding on surfaces coated by dimeric kinesin-5 (Eg5) motors (6). The authors of this report attributed the short pitch to the low processivity of Eg5, arguing that during processive episodes the motor follows the protofilament axis, but when detaching generates an off-axis force leading to microtubule rotation. Considering the structure of Eg5 (neck linker length of 18 amino acids (12)), protofilament switching may, however, also be possible during the processive episodes. For kinesin-2 (neck linker length of 17 amino acids, although reduced in length by ∼5 Å due to proline in cis-conformation at position 13 (12,13)), the propensity to switch protofilaments is controversially discussed and may depend on the stability of the neck domain (11,14).Previously, Kip3, has been found to depolymerize microtubules in a length-dependent manner (7). The underlying mechanism has been described by an antenna model, where Kip3 binds along the entire microtubule lattice and subsequently walks to the microtubule plus-end relying on its high processivity that is ∼20 times the run length of kinesin-1. During such long runs, motors in vivo are expected to frequently encounter obstacles, such as microtubule-associated proteins. In the case of kinesin-1, shown to follow the microtubule''s protofilament axis (1), obstacles cause motor stalling or accelerated detachment. It is exciting to speculate that Kip3 uses protofilament switching to bypass obstacles on the microtubule surface avoiding premature motor release or stalling that could reduce the efficiency of targeting and subsequent depolymerization of the microtubule plus-ends.     

GGA1-mediated endocytic traffic of LR11/SorLA alters APP intracellular distribution and amyloid-β production

Proteolytic processing of the amyloid-β precursor protein (APP) and generation of amyloid-β peptide (Aβ) are key events in Alzheimer's disease (AD) pathogenesis. Cell biological and genetic evidence has implicated the low-density lipoprotein and sorting receptor LR11/SorLA in AD through mechanisms related to APP and Aβ production. Defining the cellular pathway(s) by which LR11 modulates Aβ production is critical to understanding how changes in LR11 expression affect the development of Aβ pathology in AD progression. We report that the LR11 ectodomain is required for LR11-mediated reduction of Aβ and that mutagenesis of the LR11 Golgi-localizing, γ-adaptin ear homology domain, ADP-ribosylation factor (GGA)-binding motif affects the endosomal distribution of LR11, as well as LR11's effects on APP traffic and Aβ production. Targeted small interfering RNA (siRNA) knockdown studies of GGA1, GGA2, and GGA3 indicate a surprising degree of specificity toward GGA1, suggesting that GGA1 is a candidate regulator of LR11 traffic. Additional siRNA knockdown experiments reveal that GGA1 is necessary for both LR11 and β-site APP-cleaving enzyme-1 (BACE1) modulation of APP processing to Aβ. Mutagenesis of BACE1 serine 498 to alanine enhances BACE1 targeting to LR11-positive compartments and nullifies LR11-mediated reduction of Aβ. On basis of these results, we propose that GGA1 facilitates LR11 endocytic traffic and that LR11 modulates Aβ levels by promoting APP traffic to the endocytic recycling compartment.