Short Lives with Long-Lasting Effects: Filopodia Protrusions in Neuronal Branching Morphogenesis

The branching behaviors of both dendrites and axons are part of a neuronal maturation process initiated by the generation of small and transient membrane protrusions. These are highly dynamic, actin-enriched structures, collectively called filopodia, which can mature in neurons to form stable branches. Consequently, the generation of filopodia protrusions is crucial during the formation of neuronal circuits and involves the precise control of an interplay between the plasma membrane and actin dynamics. In this issue of PLOS Biology, Hou and colleagues identify a Ca²⁺/CaM-dependent molecular machinery in dendrites that ensures proper targeting of branch formation by activation of the actin nucleator Cobl.     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Dual Regulation of Dendritic Morphogenesis in Drosophila by the COP9 Signalosome

Altered dendritic arborization contributes to numerous physiological processes including synaptic plasticity, behavior, learning and memory, and is one of the most consistent neuropathologic conditions found in a number of mental retardation disorders, schizophrenia, and neurodegenerative disease. COP9 signalosome (CSN), an evolutionarily conserved regulator of the Cullin-based ubiquitin ligases that act in the proteasome pathway, has been found associated with diverse debilitating syndromes, suggesting that CSN may be involved in regulation of dendritic arborization. However, the mechanism of this control, if it exists, is unknown. To address whether the CSN pathway plays a role in dendrites, we used a simple and genetically tractable model, Drosophila larval peripheral nervous system. Our model study identified the COP9 signalosome as the key and multilayer regulator of dendritic arborization. CSN is responsible for shaping the entire dendritic tree through both stimulating and then repressing dendritic branching. We identified that CSN exerts its dualistic function via control of different Cullins. In particular, CSN stimulates dendritic branching through Cullin1, and inhibits it via control of Cullin3 function. We also identified that Cullin1 acts in neurons with the substrate-specific F-box protein Slimb to target the Cubitus interruptus protein for degradation.     

Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1^S738/742E (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1^K612W (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.     

Regulation of Neuronal Morphogenesis and Positioning by Ubiquitin-Specific Proteases in the Cerebellum

Ubiquitin signaling mechanisms play fundamental roles in the cell-intrinsic control of neuronal morphogenesis and connectivity in the brain. However, whereas specific ubiquitin ligases have been implicated in key steps of neural circuit assembly, the roles of ubiquitin-specific proteases (USPs) in the establishment of neuronal connectivity have remained unexplored. Here, we report a comprehensive analysis of USP family members in granule neuron morphogenesis and positioning in the rodent cerebellum. We identify a set of 32 USPs that are expressed in granule neurons. We also characterize the subcellular localization of the 32 USPs in granule neurons using a library of expression plasmids encoding GFP-USPs. In RNAi screens of the 32 neuronally expressed USPs, we uncover novel functions for USP1, USP4, and USP20 in the morphogenesis of granule neuron dendrites and axons and we identify a requirement for USP30 and USP33 in granule neuron migration in the rodent cerebellar cortex in vivo. These studies reveal that specific USPs with distinct spatial localizations harbor key functions in the control of neuronal morphogenesis and positioning in the mammalian cerebellum, with important implications for our understanding of the cell-intrinsic mechanisms that govern neural circuit assembly in the brain.     

Frizzled-5 Receptor Is Involved in Neuronal Polarity and Morphogenesis of Hippocampal Neurons

The Wnt signaling pathway plays important roles during different stages of neuronal development, including neuronal polarization and dendritic and axonal outgrowth. However, little is known about the identity of the Frizzled receptors mediating these processes. In the present study, we investigated the role of Frizzled-5 (Fzd5) on neuronal development in cultured Sprague-Dawley rat hippocampal neurons. We found that Fzd5 is expressed early in cultured neurons on actin-rich structures localized at minor neurites and axonal growth cones. At 4 DIV, Fzd5 polarizes towards the axon, where its expression is detected mainly at the peripheral zone of axonal growth cones, with no obvious staining at dendrites; suggesting a role of Fzd5 in neuronal polarization. Overexpression of Fzd5 during the acquisition of neuronal polarity induces mislocalization of the receptor and a loss of polarized axonal markers. Fzd5 knock-down leads to loss of axonal proteins, suggesting an impaired neuronal polarity. In contrast, overexpression of Fzd5 in neurons that are already polarized did not alter polarity, but decreased the total length of axons and increased total dendrite length and arborization. Fzd5 activated JNK in HEK293 cells and the effects triggered by Fzd5 overexpression in neurons were partially prevented by inhibition of JNK, suggesting that a non-canonical Wnt signaling mechanism might be involved. Our results suggest that, Fzd5 has a role in the establishment of neuronal polarity, and in the morphogenesis of neuronal processes, in part through the activation of the non-canonical Wnt mechanism involving JNK.     

Morphogenesis: joining the dots to shape an embryo

In the study of morphogenesis, how upstream signalling events are intricately linked to downstream cytoskeletal organisation is not entirely understood. Recent work in the Drosophila embryo has begun to shed light on this problem.     

Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.     

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.     

Dendritic nucleotides: interaction with an aliphatic acid monolayer

Dendrimeric-T and dendrimeric-A with 36 nucleotides were synthesized using phosphoramidite reagents. These dendrimers contain the nucleosides dA and dT, which are the components of the dA-dT base pair. Branching was obtained using a bifurcated and trifurcated reagent. The dendrimers were purified by ion-exchange chromatography. No specific interactions between these dendrimers were observed using atomic force microscopy. However, the dendrimeric-T is able to disrupt monolayers of fatty acids, resulting in a reorientation of the lipids (from head-to-tail to head-to-head) as demonstrated by scanning tunneling microscopy (STM).     

Morphogenesis of twisted cell wall: Chronology following an osmotic shock

Summary The effect of an osmotic shock on the subsequent growth and cell wall texture was studied at 0, 1/2, 1, 2, 4 and 24 hours. Cells were taken at the beginning of their exponential growth from mung bean hypocotyl.The shock reveals the instability and the fragility of the assembly mechanisms. It induces a rupture in the texture (formation of a loose layer) or, occasionally, the apparition of a swirling pattern. After the shock, the twisting positioning can be restored. The post-shock deposit appears similar to the pre-shock deposit. The loose layer provides a visible guide-mark (time marker) within the wall. It allows one to evaluate the oscillatory period (i.e., the duration necessary for a 180° rotation of the microfibrils). This period was found to be ca. 3 hours following a lag period of ca. 1 hour. It confirms the endogenous ultradian character of the rhythm of the assembly.