The tubulin repertoire of C. elegans sensory neurons and its context-dependent role in process outgrowth

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division (Kapitein and Hoogenraad, 2015). They are comprised of αβ‐tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms (Gogonea et al., 1999), and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code (Fulton and Simpson, 1976). Here, we use the well‐characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question, through genetic dissection of process outgrowth both in vivo and in vitro. With single‐cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons, and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba‐1, tba‐2, tbb‐1, and tbb‐2) function partially or fully redundantly, while two others (mec‐7 and mec‐12) perform specialized, context‐dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies between cell types and in vivo and in vitro contexts

Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node

The sympathetic nervous system increases heart rate by activating β adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (If) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide–sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. β adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of β adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of If. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate

Phospholipids that Contain Polyunsaturated Fatty Acids Enhance Neuronal Cell Mechanics and Touch Sensation

SummaryMechanoelectrical transduction (MeT) channels embedded in neuronal cell membranes are essential for touch and proprioception. Little is understood about the interplay between native MeT channels and membrane phospholipids, in part because few techniques are available for altering plasma membrane composition in vivo. Here, we leverage genetic dissection, chemical complementation, and optogenetics to establish that arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, enhances touch sensation and mechanoelectrical transduction activity while incorporated into membrane phospholipids in C. elegans touch receptor neurons (TRNs). Because dynamic force spectroscopy reveals that AA modulates the mechanical properties of TRN plasma membranes, we propose that this polyunsaturated fatty acid (PUFA) is needed for MeT channel activity. These findings establish that polyunsaturated phospholipids are crucial determinants of both the biochemistry and mechanics of mechanoreceptor neurons and reinforce the idea that sensory mechanotransduction in animals relies on a cellular machine composed of both proteins and membrane lipids

The tubulin repertoire of<i>C. elegans</i>sensory neurons and its context dependent role in process outgrowth

AbstractMicrotubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division (Kapitein and Hoogenraad, 2015). They are comprised of αβ-tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms (Gogoneaet al., 1999), and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code (Fulton and Simpson, 1976). Here, we use the well-characterized touch receptor neurons (TRNs) ofCaenorhabditis elegansto investigate this question, through genetic dissection of process outgrowth bothin vivoandin vitro. With single-cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons, and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba-1,tba-2,tbb-1, andtbb-2) function partially or fully redundantly, while two others (mec-7andmec-12) perform specialized, context-dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies between cell types andin vivoandin vitrocontexts.Highlight SummaryMicrotubules contribute to key cellular processes and are composed of αβ-tubulin heterodimers. Neurons inC. elegansexpress cell type-specific isoforms in addition to a shared repertoire and rely on tubulins for neurite outgrowth. Isoform function varies betweenin vivoandin vitrocontexts.AbbreviationsTRNsTouch Receptor NeuronsRNA-seqRNA sequencingRFGReceptive Field GapTPMTranscripts per MillionECMExtracellular Matrix,CVCoefficient of VariationConflict of InterestThe authors declare no conflicting financial interests.</jats:sec

The Tubulin Repertoire of Caenorhabditis Elegans Sensory Neurons and Its Context-Dependent Role in Process Outgrowth

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division. They comprise αβ-Tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms, and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code. Here we use the well-characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question through genetic dissection of process outgrowth both in vivo and in vitro. With single-cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba-1, tba-2, tbb-1, and tbb-2) function partially or fully redundantly, whereas two others (mec-7 and mec-12) perform specialized, context-dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies among cell types and in vivo and in vitro contexts

The Tubulin Repertoire of Caenorhabditis Elegans Sensory Neurons and Its Context-Dependent Role in Process Outgrowth

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division. They comprise αβ-Tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms, and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code. Here we use the well-characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question through genetic dissection of process outgrowth both in vivo and in vitro. With single-cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba-1, tba-2, tbb-1, and tbb-2) function partially or fully redundantly, whereas two others (mec-7 and mec-12) perform specialized, context-dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies among cell types and in vivo and in vitro contexts