Structural mechanism for bi-directional actin crosslinking by T-plastin

To fulfill the cytoskeleton's diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction.

Microtubule dynamics influence the retrograde biased motility of kinesin-4 motor teams in neuronal dendrites

Microtubules establish the directionality of intracellular transport by kinesins and dynein through polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus-end directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide-independent microtubule binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons induces unidirectional transport in axons but bi-directional transport with a net retrograde bias in dendrites. Removal of the secondary microtubule binding regions of KIF21B or dampening of microtubule dynamics with low concentrations of nocodazole eliminates retrograde bias in live dendrites. Further exploration of the contribution of microtubule dynamics in dendrites to directionality revealed plus-end-out microtubules to be more dynamic than plus-end-in microtubules, with nocodazole preferentially stabilizing the plus-end-out population. We propose a model in which both nucleotide-sensitive and insensitive microtubule binding sites of KIF21B motors contribute to the search and selection of stable plus-end-in microtubules within the mixed polarity microtubule arrays characteristic of mammalian dendrites to achieve net retrograde movement of KIF21B-bound cargos. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

The Finnish exclusive-negative construction ei…ku(i)n in the network of exclusion expressions

This paper shows that Finnish has the option of expressing ‘only’-like exclusive meaning with a clausal construction, here called the Exclusive-Negative Construction, ENC. It is similar to the better-known French ne…que construction but differs from it at many points where Finnish and French grammar differ, especially in the way negation is expressed in the two languages. The Finnish ENC contains overt negation, which accounts for its somewhat narrower distribution than the alternative option, the use of the focus particle vain/vaan ‘only’. Adopting the Construction Grammar framework, the paper argues that the ENC is a simple clausal construction with the idiosyncrasy of mixed polarity, a formal division of the clause into a negative and affirmative part. Derivation from an underlying comparative is rejected, but it is shown that the ENC has associations to more explicit ways of expressing exclusion. These conclusions receive support from dialectal and other non-standard data. Kokkuvõte. Maria Vilkuna: Soome keele välistav-eitav konstruktsioon ei...ku(i)n välistust väljendavate keeleliste vahendite võrgustikus. Artiklis näidatakse, et soome keeles võib väljendada välistavat tähendust (nagu ainult) lausekonstruktsiooniga, mida artiklis nimetatakse välistavaks-eitavaks konstruktsiooniks (the Exclusive-Negative Construction, ENC). See sarnaneb prantsuse keele konstruktsiooniga ne...que, ent erineb sellest mitmes aspektis, kus soome ja prantsuse keel üldiseltki erinevad, eriti mis puudutab eituse väljendamise viise kahes keeles. Soome konstruktsioon sisaldab eksplitsiitset eitust, mis seletab seda, miks konstruktsiooni kasutusala on mõnevõrra kitsam kui selle alternatiivil, fookuspartiklil vain/vaan ‘ainult’. Kasutades konstruktsioonigrammatika raamistikku, näidatakse artiklis, et ENC on lausekonstruktsioon, mille eripäraks on kahetine polaarsus: konstruktsioon jaotub vormiliselt eitavaks ja jaatavaks osaks. Väidetakse, et konstruktsioon ei ole tuletatud komparatiivikonstruktsioonist, kuid on seotud eksplitsiitsemalt välistust väljendavate vahenditega. Neid järeldusi toetavad murrete ja muu mittestandardse keelekasutuse andmed.

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior–posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.

CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells

Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.

Kinesin-4 Motor Teams Effectively Navigate Dendritic Microtubule Arrays Through Track Switching and Regulation of Microtubule Dynamics

Microtubules establish the directionality of intracellular transport by kinesins and dynein through their polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus-end directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide-independent microtubule binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons resulted in unidirectional transport in axons but bi-directional transport with a net retrograde bias in dendrites; microtubule dynamics and the secondary microtubule binding regions are required for this net directional bias. We propose a model in which cargo-bound KIF21B motors coordinate nucleotide-sensitive and insensitive microtubule binding sites to achieve net retrograde movement along the dynamic mixed polarity microtubule arrays of dendrites.

Influence of Si Substrate Preparation Procedure on Polarity of Self-Assembled GaN Nanowires on Si(111): Kelvin Probe Force Microscopy Studies

The growth of GaN nanowires having a polar, wurtzite structure on nonpolar Si substrates raises the issue of GaN nanowire polarity. Depending on the growth procedure, coexistence of nanowires with different polarities inside one ensemble has been reported. Since polarity affects the optical and electronic properties of nanowires, reliable methods for its control are needed. In this work, we use Kelvin probe force microscopy to assess the polarity of GaN nanowires grown by plasma-assisted Molecular Beam Epitaxy on Si(111) substrates. We show that uniformity of the polarity of GaN nanowires critically depends on substrate processing prior to the growth. Nearly 18% of nanowires with reversed polarity (i.e., Ga-polar) were found on the HF-etched substrates with hydrogen surface passivation. Alternative Si substrate treatment steps (RCA etching, Ga-triggered deoxidation) were tested. However, the best results, i.e., purely N-polar ensemble of nanowires, were obtained on Si wafers thermally deoxidized in the growth chamber at ~1000 °C. Interestingly, no mixed polarity was found for GaN nanowires grown under similar conditions on Si(111) substrates with a thin AlOy buffer layer. Our results show that reversal of nanowires’ polarity can be prevented by growing them on a chemically uniform substrate surface, in our case on clean, in situ formed SiNx or ex situ deposited AlOy buffers.

Talin-activated vinculin interacts with branched actin networks to initiate bundles

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.