Imaging Dendritic Spines in <em>Caenorhabditis elegans</em>

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Vanderbilt University

 is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive  alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of  growth defects yielded , which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion.  suppressed the vacuole fragmentation phenotype of , but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion  and is a potential transport substrate of Neo1. Strains deficient in PE synthesis () displayed fragmented vacuoles and the  fragmented vacuole phenotype was also suppressed by overexpression of , encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast,  was not suppressed by overexpression of , an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane.

Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in .

GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand ( ) at the genomic locus of each target protein. The resultant  knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment () in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the  nervous system.

NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in .

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in  (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that  motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of  genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.

 neurons have functional dendritic spines.

Sydney Brenner's choice of  as a model organism for understanding the nervous system has accelerated discoveries of gene function in neural circuit development and behavior. In this review, we discuss a striking example of synaptic remodeling in the  motor circuit in which DD class motor neurons effectively reverse polarity as presynaptic and postsynaptic domains at opposite ends of the DD neurite switch locations. Originally revealed by EM reconstruction conducted over 40 years ago, DD remodeling has since been investigated by live cell imaging methods that exploit the power of  genetics to reveal key effectors of synaptic plasticity. Although synapses are also extensively rewired in developing mammalian circuits, the underlying remodeling mechanisms are largely unknown. Here, we highlight the possibility that studies in  can reveal pathways that orchestrate synaptic remodeling in more complex organisms. Specifically, we describe (1) transcription factors that regulate DD remodeling, (2) the cellular and molecular cascades that drive synaptic remodeling and (3) examples of circuit modifications in vertebrate neurons that share some similarities with synaptic remodeling in  DD neurons.

Synaptic remodeling, lessons from .

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel ENaC/UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-α/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in  (Miller-Fleming et al., 2016). Here, we report that the conserved transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination during development and reveal parallel-acting pathways that coordinate synaptic disassembly by removing specific active zone proteins.Synaptic pruning is a conserved feature of developing neural circuits but the mechanisms that dismantle the presynaptic apparatus are largely unknown. We have determined that synaptic disassembly is orchestrated by parallel-acting mechanisms that target distinct components of the active zone. Thus, our finding suggests that synaptic disassembly is not accomplished by en masse destruction but depends on mechanisms that dismantle the structure in an organized process.

Andrea Cuentas-Condori

Department of Cell and Developmental Biology

Vanderbilt University

Imaging Dendritic Spines in Caenorhabditis elegans

Andrea Cuentas-Condori

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Developmental Biology

Vanderbilt University

Imaging Dendritic Spines in Caenorhabditis elegans

Department of Cell and Developmental Biology