RESUMEN
The polarized nature of neurons depends on their microtubule dynamics and orientation determined by both microtubule-stabilizing and destabilizing factors. The role of destabilizing factors in developing and maintaining neuronal polarity is unclear. We investigated the function of KLP-7, a microtubule depolymerizing motor of the Kinesin-13 family, in axon-dendrite compartmentalization using PVD neurons in Caenorhabditis elegans. Loss of KLP-7 caused a mislocalization of axonal proteins, including RAB-3, SAD-1, and their motor UNC-104, to dendrites. This is rescued by cell-autonomous expression of the KLP-7 or colchicine treatment, indicating the involvement of KLP-7-dependent microtubule depolymerization. The high mobility of KLP-7 is correlated to increased microtubule dynamics in the dendrites, which restricts the enrichment of UNC-44, an integral component of Axon Initial Segment (AIS) in these processes. Due to the loss of KLP-7, ectopic enrichment of UNC-44 in the dendrite potentially redirects axonal traffic into dendrites that include plus-end out microtubules, axonal motors, and cargoes. These observations indicate that KLP-7-mediated depolymerization defines the microtubule dynamics conducive to the specific enrichment of AIS components in dendrites. This further compartmentalizes dendritic and axonal microtubules, motors, and cargoes, thereby influencing neuronal polarity.
Asunto(s)
Axones , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Polaridad Celular , Dendritas , Cinesinas , Microtúbulos , Animales , Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Axones/metabolismo , Polaridad Celular/fisiología , Neuronas/metabolismo , Transporte de Proteínas , Proteínas del Tejido Nervioso/metabolismoRESUMEN
PVD neuron of Caenorhabditis elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities including the control of both nociceptive touch sensation and body posture. Although both the axon and dendrites of this neuron show a regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1/MLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration through the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery in posture. Our data revealed that the axons and dendrites of PVD neurons regulate the nociception and proprioception in worms, respectively. It also revealed that dendrite and axon regeneration lead to the restoration of these differential sensory modalities.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Dendritas , Regeneración Nerviosa , Animales , Caenorhabditis elegans/fisiología , Dendritas/fisiología , Regeneración Nerviosa/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Recuperación de la Función/fisiología , Células Receptoras Sensoriales/fisiología , Axotomía , Tacto/fisiología , Animales Modificados Genéticamente , Axones/fisiología , Quinasas Quinasa Quinasa PAMRESUMEN
PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to its elaborate and stereotype dendrite morphology. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to knocking down genes in nervous system. In order to optimize the RNAi conditions for PVD neuron, we fed the worm strains with E. coli HT115 bacteria expressing dsRNA against mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the available sensitive backgrounds including the one expresses sid-1 under unc-119 promoter, although resulted in reduction of dendrite branching, the phenotypes were significantly modest compared to the respective loss of function mutants. In order to enhance RNAi in PVD neurons, we generated a strain that expressed sid-1 under the promoter mec-3, which exhibits strong expression in PVD. When Pmec-3::sid-1 is expressed in either nre-1(-)lin-15b(-) or lin-15b(-) backgrounds, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to the genetic background alone. Moreover, knockdown of genes playing role in dendrite regeneration in the nre-1(-)lin-15b(-), Pmec-3-sid-1[+] background resulted in significant reduction in dendrite regeneration following laser injury. The extent of dendrite regrowth due to the RNAi of aff-1 or ced-10 in our optimized strain was comparable to that of aff-1 and ced-10 mutants. Essentially, our strain expressing sid-1 in PVD neuron, provides an RNAi optimized platform for high throughput screening of genes involved in PVD development, maintenance and regeneration.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Interferencia de ARN , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Neuronas/metabolismoRESUMEN
Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and reduction of gentle touch sensation in mbl-1 mutant. Our data also revealed that mbl-1 is genetically epistatic to mec-7 (ß tubulin) and mec-12 (α tubulin) in regulating axon growth. Furthermore, mbl-1 is epistatic to sad-1, an ortholog of BRSK/Brain specific-serine/threonine kinase and a known regulator of synaptic machinery, for synapse formation at the correct location of the PLM neurite. Notably, the immunoprecipitation of MBL-1 resulted in the co-purification of mec-7, mec-12, and sad-1 mRNAs, suggesting a direct interaction between MBL-1 and these transcripts. Additionally, mbl-1 mutants exhibited reduced levels and stability of mec-7 and mec-12 transcripts. Our study establishes a previously unknown link between RNA-binding proteins and cytoskeletal machinery, highlighting their crucial roles in the development and maintenance of the nervous system.
Asunto(s)
Caenorhabditis elegans , Tubulina (Proteína) , Animales , Tubulina (Proteína)/genética , Caenorhabditis elegans/genética , ARN Mensajero , Citoesqueleto/genética , Microtúbulos/genética , NeuronasRESUMEN
Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects-territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas de Unión al GTP rac , Animales , Axones/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Regeneración Nerviosa/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.
Asunto(s)
Axones , Caenorhabditis elegans , Animales , Axones/fisiología , Dendritas/fisiología , Microtúbulos/fisiología , Regeneración Nerviosa , NeuronasRESUMEN
Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16's inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Regeneración Nerviosa , Neuronas/fisiología , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Microtúbulos/metabolismo , Modelos Animales , Mutación , Isoformas de Proteínas/metabolismo , Análisis Espacio-TemporalRESUMEN
The adult nervous system has a limited capacity to regenerate after accidental damage. Post-injury functional restoration requires proper targeting of the injured axon to its postsynaptic cell. Although the initial response to axonal injury has been studied in great detail, it is rather unclear what controls the re-establishment of a functional connection. Using the posterior lateral microtubule neuron in Caenorhabditis elegans, we found that after axotomy, the regrowth from the proximal stump towards the ventral side and accumulation of presynaptic machinery along the ventral nerve cord correlated to the functional recovery. We found that the loss of insulin receptor DAF-2 promoted 'ventral targeting' in a DAF-16-dependent manner. We further showed that coordinated activities of DAF-16 in neuron and muscle promoted 'ventral targeting'. In response to axotomy, expression of the Netrin receptor UNC-40 was upregulated in the injured neuron in a DAF-16-dependent manner. In contrast, the DAF-2-DAF-16 axis contributed to the age-related decline in Netrin expression in muscle. Therefore, our study revealed an important role for insulin signaling in regulating the axon guidance molecules during the functional rewiring process.
Asunto(s)
Axones/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Netrinas/metabolismo , Animales , Orientación del Axón , Proteínas de Caenorhabditis elegans/genética , Moléculas de Adhesión Celular/genética , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Microtúbulos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Netrina/metabolismo , Netrinas/genética , Neuronas/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Neurogénesis/genética , Vía de Señalización Wnt/genética , Animales , Axones/metabolismo , Axones/ultraestructura , Transporte Biológico , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciación Celular , Polaridad Celular/genética , Dendritas/metabolismo , Dendritas/ultraestructura , Regulación de la Expresión Génica , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Restoration of lost function following a nervous system injury is limited in adulthood as the regenerative capacity of nervous system declines with age. Pharmacological approaches have not been very successful in alleviating the consequences of nervous system injury. On the contrary, physical activity and rehabilitation interventions are often beneficial to improve the health conditions in the patients with neuronal injuries. Using touch neuron circuit of Caenorhabditis elegans, we investigated the role of physical exercise in the improvement of functional restoration after axotomy. We found that a swimming session of 90 min following the axotomy of posterior lateral microtubule (PLM) neuron can improve functional recovery in larval and adult stage animals. In older age, multiple exercise sessions were required to enhance the functional recovery. Genetic analysis of axon regeneration mutants showed that exercise-mediated enhancement of functional recovery depends on the ability of axon to regenerate. Exercise promotes early initiation of regrowth, self-fusion of proximal and distal ends, as well as postregrowth enhancement of function. We further found that the swimming exercise promotes axon regeneration through the activity of cellular energy sensor AAK-2/AMPK in both muscle and neuron. Our study established a paradigm where systemic effects of exercise on functional regeneration could be addressed at the single neuron level.
Asunto(s)
Axones , Proteínas de Caenorhabditis elegans , Proteínas Quinasas Activadas por AMP , Adulto , Anciano , Animales , Proteínas de Caenorhabditis elegans/genética , Humanos , Regeneración Nerviosa , NataciónRESUMEN
Ethanol is a widely used drug, excessive consumption of which could lead to medical conditions with diverse symptoms. Ethanol abuse causes dysfunction of memory, attention, speech and locomotion across species. Dopamine signaling plays an essential role in ethanol dependent behaviors in animals ranging from C. elegans to humans. We devised an ethanol dependent assay in which mutants in the dopamine autoreceptor, dop-2, displayed a unique sedative locomotory behavior causing the animals to move in circles while dragging the posterior half of their body. Here, we identify the posterior dopaminergic sensory neuron as being essential to modulate this behavior. We further demonstrate that in dop-2 mutants, ethanol exposure increases dopamine secretion and functions in a DVA interneuron dependent manner. DVA releases the neuropeptide NLP-12 that is known to function through cholinergic motor neurons and affect movement. Thus, DOP-2 modulates dopamine levels at the synapse and regulates alcohol induced movement through NLP-12.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Etanol/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Depresores del Sistema Nervioso Central/farmacología , Dopamina/metabolismo , Neuronas Dopaminérgicas/fisiología , Humanos , Locomoción/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Mutación , Neuropéptidos/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
The G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) in the olfactory system function to sense the surrounding environment and respond to various odorants. The genes coding for olfactory receptors in Caenorhabditis elegans are larger in number in comparison to those in mammals, suggesting complexity in the receptor-odorant relationships. Recent studies have shown that the same odorant in different concentrations could act on multiple receptors in different neurons to induce attractive or repulsive responses. The ASH neurons are known to be responsible for responding to high concentrations of volatile odorants. Here, we characterize a new GPCR, SRX-97. We found that the srx-97 promoter drives expression specifically in the head ASH and tail PHB chemosensory neurons of C. elegans Moreover, the SRX-97 protein localizes to the ciliary ends of the ASH neurons. Analysis of clustered regularly interspaced short palindromic repeats (CRISPR)-based deletion mutants of the srx-97 locus suggests that this gene is involved in recognition of high concentrations of benzaldehyde. This was further confirmed through rescue and neuronal ablation experiments. Our work brings novel insights into concentration-dependent receptor function in the olfactory system, and provides details of an additional molecule that helps the animal navigate its surroundings.
Asunto(s)
Neuronas Receptoras Olfatorias , Receptores Odorantes , Animales , Benzaldehídos , Caenorhabditis elegans , Odorantes , Receptores Odorantes/genéticaRESUMEN
Neuronal injury often leads to devastating consequences such as loss of senses or locomotion. Restoration of function after injury relies on whether the injured axons can find their target cells. Although fusion between injured proximal axon and distal fragment has been observed in many organisms, its functional significance is not clear. Here, using Caenorhabditis elegans mechanosensory neurons, we address this question. Using two femtosecond lasers simultaneously, we could scan and sever posterior lateral microtubule neurons [posterior lateral microtubules (PLMs)] on both sides of the worm. We showed that axotomy of both PLMs leads to a dramatic loss of posterior touch sensation. During the regenerative phase, only axons that fuse to their distal counterparts contribute to functional recovery. Loss of let-7 miRNA promotes functional restoration in both larval and adult stages. In the L4 stage, loss of let-7 increases fusion events by increasing the mRNA level of one of the cell-recognition molecules, CED-7. The ability to establish cytoplasmic continuity between the proximal and distal ends declines with age. Loss of let-7 overcomes this barrier by promoting axonal transport and enrichment of the EFF-1 fusogen at the growing tip of cut processes. Our data reveal the functional property of a regenerating neuron.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Axones/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Glicoproteínas de Membrana/fisiología , MicroARNs/metabolismo , Regeneración Nerviosa/genética , Células Receptoras Sensoriales/fisiología , Animales , Transporte Axonal/fisiología , Axotomía , Citoplasma/fisiología , Microtúbulos/fisiología , TactoRESUMEN
The microtubule (MT) cytoskeleton of a mature axon is maintained in a stabilized steady state, yet after axonal injury it can be transformed into a dynamic structure capable of supporting axon regrowth. Using Caenorhabditis elegans mechanosensory axons and in vivo imaging, we find that, in mature axons, the growth of MTs is restricted in the steady state by the depolymerizing kinesin-13 family member KLP-7. After axon injury, we observe a two-phase process of MT growth upregulation. First, the number of growing MTs increases at the injury site, concomitant with local downregulation of KLP-7. A second phase of persistent MT growth requires the cytosolic carboxypeptidase CCPP-6, which promotes Δ2 modification of α-tubulin. Both phases of MT growth are coordinated by the DLK-1 MAP kinase cascade. Our results define how the stable MT cytoskeleton of a mature neuron is converted into the dynamically growing MT cytoskeleton of a regrowing axon.
Asunto(s)
Axones/metabolismo , Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Regeneración Nerviosa , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Animales , Caenorhabditis elegans/genéticaRESUMEN
The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and in vivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.
Asunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Pruebas Genéticas/métodos , Regeneración Nerviosa/fisiología , Animales , Caenorhabditis elegans/anatomía & histología , Proteínas de Caenorhabditis elegans/metabolismo , Endocitosis , Familia de Multigenes , Mutación , Vesículas Sinápticas/metabolismoRESUMEN
Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-ßPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Dineínas/metabolismo , Regulación de la Expresión Génica , Espermatogonias/fisiología , Alelos , Animales , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Dineínas/genética , Integrina beta1/metabolismo , Masculino , Mutación , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Fenotipo , Testículo/fisiología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Axons of adult Caenorhabditis elegans neurons undergo robust regenerative growth after laser axotomy. Here we show that axotomy of PLM sensory neurons triggers axonal calcium waves whose amplitude correlates with the extent of regeneration. Genetic elevation of Ca(2+) or cAMP accelerates formation of a growth cone from the injured axon. Elevated Ca(2+) or cAMP also facilitates apparent fusion of axonal fragments and promotes branching to postsynaptic targets. Conversely, inhibition of voltage-gated calcium channels or calcium release from internal stores reduces regenerative growth. We identify the fusogen EFF-1 as critical for axon fragment fusion and the basic leucine zipper domain (bZip) protein CREB (cAMP response element-binding protein) as a key effector for branching. The effects of elevated Ca(2+) or cAMP on regrowth require the MAPKKK (mitogen-activated protein kinase kinase kinase) DLK-1. Increased cAMP signaling can partly bypass the requirement for the bZip protein CEBP-1, a downstream factor of the DLK-1 kinase cascade. These findings reveal the relationship between Ca(2+)/cAMP signaling and the DLK-1 MAPK (mitogen-activated protein kinase) cascade in regeneration.
Asunto(s)
Axones/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Regeneración Nerviosa/fisiología , Animales , Axotomía , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Conos de Crecimiento/metabolismo , Rayos Láser , Sistema de Señalización de MAP Quinasas/fisiología , Glicoproteínas de Membrana/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.
Asunto(s)
Dineínas Citoplasmáticas/metabolismo , Dineínas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Dineínas Citoplasmáticas/genética , Dineínas/genética , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ligandos , Lisosomas/metabolismo , Multimerización de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Axonal regeneration in Caenorhabditis elegans was first reported five years ago. Individual green fluorescent protein-labeled axons can be severed using laser microsurgery and their regrowth followed in vivo. Several neuron types display robust regrowth after injury, including motor and sensory neurons. The small size and transparency of C. elegans make possible large-scale genetic and pharmacological screens for regeneration phenotypes.