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Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5β (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid–liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mechanism that regulates dendritic microtubule organization is still unclear. Cao et al. demonstrate that the microtubule minus-end binding protein CAMSAP2 and kinesin-14 motor KIFC3 work together to organize dendritic microtubules and control dendrite branching.

Summary Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites., Graphical Abstract, Highlights • KIFC3 localizes to dendrites and controls dendrite branching • KIFC3 interacts with minus-end binding protein CAMSAP2 • CAMSAP2 and KIFC3 immobilize microtubule arrays in dendrites, Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mechanism that regulates dendritic microtubule organization is still unclear. Cao et al. demonstrate that the microtubule minus-end binding protein CAMSAP2 and kinesin-14 motor KIFC3 work together to organize dendritic microtubules and control dendrite branching.

Additional file 1: Fig. S1. Bicd2 is expressed by RGPs and post-mitotic neurons in the cortex of control mice, but abolished after cell-type-specific depletion of BICD2 in cKO mice. a. Coronal cryo-sections of E17.5 brains from cell-type-specific conditional Bicd2 KO and control mice - Bicd2fl/fl;Nex-Cre+/− (=Nex-KO), Bicd2fl/fl;Emx1-Cre+/− (=Emx1-KO) and Bicd2fl/fl;Cre−/− (=Nex-WT/Emx1-WT) respectively - were stained against BICD2. Scale bars are 500 μm. b. Coronal cryo-sections of E17.5 cortices from Nex-KO, Emx1-KO and Nex-WT/Emx1-WT mice were stained against BICD2 (red). DAPI is shown in blue. Scale bars are 100 μm. c. Zooms from BICD2 staining shown in (b). d + e. Western Blots (WBs) of whole E17.5 cortex lysates from Nex-KO, Emx1-KO, and their control littermates Nex-WT and Emx1-WT. WBs were stained with commercial anti-BICD2 (atlas), home-made anti- BICD2 (#2294), home-made anti-BICD1/2(#2293), anti-actin or anti-tubulin. M = marker. Molecular weight of the marker bands are indicated on the left in kDa. (N = 3). f. Radial diameter of the cortex from ventricular to pial surface of Nex-WT, Nex-KO, Emx1-WT and Emx1-KO mice (in μm) (N = 16–17). *** p

Summary The motor protein kinesin-1 plays an important role in polarized sorting of transport vesicles to the axon. However, the mechanism by which the axonal entry of kinesin-1-dependent cargo transport is regulated remains unclear. Microtubule-associated protein MAP7 (ensconsin in Drosophila) is an essential kinesin-1 cofactor and promotes kinesin-1 recruitment to microtubules. Here, we found that MAP7 family member MAP7D2 concentrates at the proximal axon, where it overlaps with the axon initial segment and interacts with kinesin-1. Depletion of MAP7D2 results in reduced axonal cargo entry and defects in axon development and neuronal migration. We propose a model in which MAP7D2 in the proximal axon locally promotes kinesin-1-mediated cargo entry into the axon., Graphical Abstract, Highlights • The microtubule-binding domain of MAP7D2 targets the proximal axon • MAP7D2 is required for axon development • MAP7D2 associates with all three kinesin-1 family members • MAP7D2 promotes kinesin-1-mediated cargo transport in axons, Kinesin-1 is well known for transporting vesicles and organelles into to the axon. Pan et al. show that MAP7D2 concentrates at the proximal axon, regulates kinesin-1 activity, and locally promotes kinesin-1-mediated cargo trafficking into axons. Specific microtubule-associated proteins (MAPs) that locally control motor activities is an emerging concept.

Mixed polarity microtubule organization is the signature characteristic of vertebrate dendrites. Oppositely oriented microtubules form the basis for selective cargo trafficking in neurons, however the mechanisms that establish and maintain this organization are unclear. Here, we show that APC2, the brain-specific homolog of tumor-suppressor protein adenomatous polyposis coli (APC), promotes dynamics of minus-end-out microtubules in dendrites. We found that APC2 localizes as distinct clusters along microtubule bundles in dendrites and that this localization is driven by LC8-binding and two separate microtubule-interacting domains. Depletion of APC2 reduces the plus end dynamics of minus-end-out oriented microtubules, increases microtubule sliding, and causes defects in dendritic morphology. We propose a model in which APC2 regulates dendrite development by promoting dynamics of minus-end-out microtubules., Microtubules in dendrites are characterized by mixed polarity orientation. Here, the authors show a role for adenomatous polyposis coli 2 (APC2) in regulating dendrite microtubule dynamics and dendrite development.

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity. Farías et al. report that the localization of the endoplasmic reticulum (ER) in the axon is controlled by the interaction between ER-shaping proteins and the microtubule cytoskeleton. Local ER and microtubule crosstalk promotes ER tubule-microtubule stabilization and drives neuronal polarity.