RESUMO
Lysosomes, single-membrane organelles defined by a uniquely strong acidic lumenal pH and high content of acid hydrolases, are the shared degradative compartments of the endocytic and autophagic pathways. These pathways, and especially lysosomes, are points of particular vulnerability in many neurodegenerative diseases. Beyond the role of lysosomes in substrate degradation, new findings have ascribed to lysosomes the leading role in sensing and responding to cellular nutrients, growth factors and cellular stress. This review aims to integrate recent concepts of basic lysosome biology and pathobiology as a basis for understanding neurodegenerative disease pathogenesis. Here, we discuss the newly recognized signaling functions of lysosomes and specific aspects of lysosome biology in neurons while re-visiting the classical defining criteria for lysosomes and the importance of preserving strict definitions. Our discussion emphasizes dynein-mediated axonal transport of maturing degradative organelles, with further consideration of their roles in synaptic function. We finally examine how distinctive underlying disturbances of lysosomes in various neurodegenerative diseases result in unique patterns of auto/endolysosomal mistrafficking. The rapidly emerging understanding of lysosomal trafficking and disruptions in lysosome signaling is providing valuable clues to new targets for disease-modifying therapies.
Assuntos
Lisossomos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Animais , HumanosRESUMO
Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr(407) and p-FAK-Tyr(397) display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr(407) and Tyr(397). Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr(407) phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.
Assuntos
Barreira Hematotesticular/enzimologia , Quinase 1 de Adesão Focal/metabolismo , Células de Sertoli/metabolismo , Junções Íntimas/enzimologia , Actinas/genética , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Barreira Hematotesticular/citologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/genética , Multimerização Proteica/fisiologia , Ratos , Células de Sertoli/citologia , Espermatócitos/citologia , Espermatócitos/enzimologia , Espermatogênese/fisiologia , Junções Íntimas/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Throughout spermatogenesis, a select pool of germ cells, the leptotene spermatocytes, must traverse the blood-testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium. This event requires extensive restructuring of cell junctions, and it must also coincide with germ cell cycle progression in preparation for primary spermatocyte meiosis. Recent findings show that cell-cycle-associated kinases and phosphatases, including mitogen-activated protein kinases (MAPKs), participate in the pathways that also direct germ cell adhesion and movement. Our new biochemical model explains, in part, how two distinct cellular events, BTB restructuring and spermiation, are coordinated to maintain spermatogenesis and fertility. In this way, MAPKs would synchronize cell cycle progression in primary spermatocytes with junction remodeling and cell migration across the BTB.
Assuntos
Barreira Hematotesticular/fisiologia , Junções Intercelulares/fisiologia , Espermatogênese/fisiologia , Animais , Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Desmossomos/fisiologia , Humanos , Masculino , Prófase Meiótica I/fisiologia , Modelos Biológicos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismoRESUMO
Interleukin (IL)-1 is a proinflammatory cytokine with important roles in innate immunity, as well as in normal tissue homeostasis. Interestingly, recent studies have also shown IL-1 to function in the dynamics of the actin cytoskeleton and cell junctions. For example, treatment of different epithelia with IL-1α often results in the restructuring of the actin network and cell junctions, thereby leading to junction disassembly. In this review, we highlight new and interesting findings that show IL-1 to be a critical player of restructuring events in the seminiferous epithelium of the testis during spermatogenesis.
Assuntos
Citoesqueleto de Actina/metabolismo , Junções Intercelulares/metabolismo , Interleucina-1/metabolismo , Humanos , Interleucina-1/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Espermatogênese , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood-testis barrier (BTB) must remain intact during the transit of spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with spermatid movement and proper spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of spermatid transit plus a loss of proper orientation in the epithelium, and a "tightened" Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.
Assuntos
Proteína 3 Relacionada a Actina/metabolismo , Barreira Hematotesticular/metabolismo , Espermatogênese , Junções Íntimas/metabolismo , Animais , Carbazóis/farmacologia , Comunicação Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Epitélio/patologia , Masculino , Propanolaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Células de Sertoli/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Throughout spermatogenesis, the Sertoli cell blood-testis barrier (BTB) is strictly regulated by cytokines, which mediate its timely restructuring, thereby allowing spermatocytes to enter the adluminal compartment of the seminiferous epithelium for development into spermatozoa. The aim herein was to investigate whether germ cells play a role in BTB restructuring via the action of interleukin-1α (IL-1α) since germ cells are known to control Sertoli cell production of this cytokine, and if yes, how these effects are mediated. When Sertoli cells were isolated from Sprague-Dawley rats and plated at high density, IL-1α (100 pg/ml) was shown to "open" the Sertoli cell barrier when its integrity was assessed by transepithelial electrical resistance measurements. Further investigation of Sertoli cells treated with IL-1α revealed striking changes in the cellular distribution of actin filaments when compared to untreated cells. These effects at the Sertoli cell barrier were mediated, in part, by epidermal growth factor receptor pathway substrate 8 (Eps8; an actin bundling and barbed-end capping protein) and actin-related protein 3 (Arp3; a component of the actin nucleation machinery). As important, an increase in the kinetics of occludin internalization but a decrease in its rate of degradation was noted following IL-1α treatment. These results indicate that IL-1α is a critical regulator of BTB dynamics.
Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Interleucina-1alfa/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Células Cultivadas , Endocitose/efeitos dos fármacos , Interleucina-1alfa/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1RESUMO
The blood-testis barrier (BTB), similar to other blood-tissue barriers, such as the blood-brain barrier and the blood-retinal barrier, is used to protect the corresponding organ from harmful substances (e.g., xenobiotics) including drugs and foreign compounds. More importantly, the BTB allows postmeiotic spermatid development to take place in an immune privileged site at the adluminal (or apical) compartment to avoid the production of antibodies against spermatid-specific antigens, many of which express transiently during spermiogenesis and spermiation. The BTB, however, also poses an obstacle in developing nonhormonal-based male contraceptives by sequestering drugs (e.g., adjudin) that exert their effects on germ cells in the adluminal compartment. The effects of these drugs include disruption of germ cell cycle progression and development, apoptosis, cell adhesion, metabolism and others. Recent studies have demonstrated that there is a functional axis that operates locally in the seminiferous epithelium to co-ordinate different cellular events across the Sertoli cell epithelium, such as spermiation and BTB restructuring during the seminiferous epithelial cycle of spermatogenesis. Components of this functional axis, such as the apical ectoplasmic specialization (apical ES, a testis-specific atypical anchoring junction type) and the BTB, in particular their constituent protein complexes, such as alpha6beta1-integrin and occludin at the apical ES and the BTB, respectively, can be the target of male contraception. In this chapter, we highlight recent advances regarding the likely mechanism of action of adjudin in this functional axis with emphasis on the use of molecular modeling technique to facilitate the design of better compounds in male contraceptive development.
Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Anticoncepção/métodos , Espermatogênese/efeitos dos fármacos , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Sequência de Aminoácidos , Animais , Barreira Hematotesticular/metabolismo , Domínio Catalítico , Anticoncepcionais Masculinos/metabolismo , Anticoncepcionais Masculinos/farmacologia , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacologia , Ligação de Hidrogênio , Indazóis/metabolismo , Indazóis/farmacologia , Integrina alfa6beta1/metabolismo , Masculino , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ocludina/metabolismo , Epitélio Seminífero/efeitos dos fármacos , Epitélio Seminífero/metabolismo , Alinhamento de Sequência , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismoRESUMO
Dysfunction and mistrafficking of organelles in autophagy- and endosomal-lysosomal pathways are implicated in neurodegenerative diseases. Here, we reveal selective vulnerability of maturing degradative organelles (late endosomes/amphisomes) to disease-relevant local calcium dysregulation. These organelles undergo exclusive retrograde transport in axons, with occasional pauses triggered by regulated calcium efflux from agonist-evoked transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) channels-an effect greatly exaggerated by exogenous agonist mucolipin synthetic agonist 1 (ML-SA1). Deacidification of degradative organelles, as seen after Presenilin 1 (PSEN1) loss of function, induced pathological constitutive "inside-out" TRPML1 hyperactivation, slowing their transport comparably to ML-SA1 and causing accumulation in dystrophic axons. The mechanism involved calcium-mediated c-Jun N-terminal kinase (JNK) activation, which hyperphosphorylated dynein intermediate chain (DIC), reducing dynein activity. Blocking TRPML1 activation, JNK activity, or DIC1B serine-80 phosphorylation reversed transport deficits in PSEN1 knockout neurons. Our results, including features demonstrated in Alzheimer-mutant PSEN1 knockin mice, define a mechanism linking dysfunction and mistrafficking in lysosomal pathways to neuritic dystrophy under neurodegenerative conditions.
RESUMO
Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.
Assuntos
Anticoncepcionais Masculinos/farmacologia , Hidrazinas/farmacologia , Indazóis/farmacologia , Epitélio Seminífero/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Anticoncepcionais Masculinos/toxicidade , Humanos , Hidrazinas/toxicidade , Indazóis/toxicidade , Indenos/farmacologia , Masculino , Piperidinas/farmacologia , Células de Sertoli/efeitos dos fármacosRESUMO
Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7+ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.
Assuntos
Axônios/metabolismo , Complexo de Golgi/metabolismo , Organelas/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos TransgênicosRESUMO
In the seminiferous epithelium, Eps8 is localized to actin-based cell junctions at the blood-testis barrier (BTB) and the apical ectoplasmic specialization (ES) in stage V-VI tubules but is considerably diminished in stage VIII tubules. Eps8 down-regulation coincides with the time of BTB restructuring and apical ES disassembly, implicating the role of Eps8 in cell adhesion. Its involvement in Sertoli-germ cell adhesion was substantiated in studies using an in vivo animal model by treating rats with 1-(2,4-dichlorobenzy)-1H-indazole-3-carbohydrazide (adjudin) to induce anchoring junction restructuring, during which Eps8 disappeared at the apical ES before germ cell departure. In Sertoli cell cultures with established permeability barrier mimicking the BTB in vivo, the knockdown of Eps8 by RNAi led to F-actin disorganization and the mislocalization of the tight junction proteins occludin and ZO-1, suggesting the function of Eps8 in maintaining BTB integrity. In vivo knockdown of Eps8 in the testis caused germ cell sloughing and BTB damage, concomitant with occludin mislocalization, further validating that Eps8 is a novel regulator of cell adhesion and BTB integrity in the seminiferous epithelium.
Assuntos
Barreira Hematotesticular/fisiologia , Adesão Celular/fisiologia , Proteínas/fisiologia , Epitélio Seminífero/citologia , Epitélio Seminífero/fisiologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Barreira Hematotesticular/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Primers do DNA/genética , Hidrazinas/farmacologia , Indazóis/farmacologia , Masculino , Proteínas/antagonistas & inibidores , Proteínas/genética , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/efeitos dos fármacos , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/fisiologia , Espermátides/citologia , Espermátides/efeitos dos fármacos , Espermátides/fisiologia , Espermatogênese/fisiologiaRESUMO
In adult rat testes, blood-testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient 'opening'. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), beta-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/beta-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with beta-catenin and ZO-1. Beta-catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an 'engaged'state to reinforce the barrier function in normal testes. However, beta-catenin and ZO-1 were 'disengaged' from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the 'disengagement' of beta-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.
Assuntos
Barreira Hematotesticular , Caderinas/metabolismo , Dinamina II/metabolismo , Proteínas de Membrana/metabolismo , Testículo/fisiologia , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Caderinas/análise , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Dinamina II/análise , Hidrazinas/análise , Hidrazinas/metabolismo , Hidrazinas/farmacologia , Imuno-Histoquímica/métodos , Indazóis/análise , Indazóis/metabolismo , Indazóis/farmacologia , Masculino , Proteínas de Membrana/análise , Microscopia de Fluorescência , Nectinas , Ocludina , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Espermatozoides/fisiologia , Testículo/metabolismo , Proteína da Zônula de Oclusão-1 , beta Catenina/análise , beta Catenina/metabolismoRESUMO
Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.
Assuntos
Cálcio/metabolismo , Lisossomos/metabolismo , Presenilina-1/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia , Linhagem Celular , Homeostase , Concentração de Íons de Hidrogênio , Camundongos , Presenilina-1/genética , ProteóliseRESUMO
Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-ß2, TGF-ß3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells.
Assuntos
Bioensaio , Barreira Hematotesticular/metabolismo , Endocitose/genética , Endossomos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/genética , Animais , Avidina/química , Biotina/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Masculino , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células de Sertoli/citologia , Transdução de Sinais , Testosterona/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Germ cell transport across the seminiferous epithelium during the epithelial cycle is crucial to spermatogenesis, although molecular mechanism(s) that regulate these events remain unknown. Studies have shown that spatiotemporal expression of crucial regulatory proteins during the epithelial cycle represents an efficient and physiologically important mechanism to regulate spermatogenesis without involving de novo synthesis of proteins and/or expression of genes. Herein, we critically review the role of focal adhesion kinase (FAK) in coordinating the transport of spermatids and preleptotene spermatocytes across the epithelium and the BTB, respectively, along the apical ectoplasmic specialization (ES) - blood-testis barrier - basement membrane (BM) functional axis during spermatogenesis. In the testis, p-FAK-Tyr³⁸⁷ and p-FAK-Tyr⁴⁰⁷ are spatiotemporally expressed during the epithelial cycle at the actin-rich anchoring junction known as ES, regulating cell adhesion at the Sertoli-spermatid (apical ES) and Sertoli cell-cell (basal ES) interface. Phosphorylated forms of FAK exert their effects by regulating the homeostasis of F-actin at the ES, mediated via their effects on actin polymerization so that microfilaments are efficiently re-organized, such as from their "bundled" to "de-bundled/branched" configuration and vice versa during the epithelial cycle to facilitate the transport of: (i) spermatids across the epithelium, and (ii) preleptotene spermatocytes across the BTB. In summary, p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁸⁷ are important regulators of spermatogenesis which serve as molecular switches that turn "on" and "off" adhesion function at the apical ES and the basal ES/BTB, mediated via their spatiotemporal expression during the epithelial cycle. A hypothetical model depicting the role of these two molecular switches is also proposed.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Espermatogênese/fisiologia , Animais , Humanos , MasculinoRESUMO
Throughout mammalian spermatogenesis, preleptotene/leptotene spermatocytes traverse the blood-testis barrier during stages VIII-XI of the seminiferous epithelial cycle while trapped within a dynamic intermediate compartment that is sealed at north and south poles by tight junctions, basal ectoplasmic specializations, desmosomes and gap junctions. In order for spermatocytes to gain entry into the adluminal compartment of the seminiferous epithelium for continued development, 'old' junctions present above migrating spermatocytes disassemble, while 'new' junctions assemble simultaneously below these germ cells. In this way, the integrity of the blood-testis barrier and the homeostasis of the seminiferous epithelium can remain intact during spermatogenesis. Previous studies have shown an array of cellular events, including protein internalization and cytoskeletal remodeling, to underline blood-testis barrier restructuring, whereas other studies have reported BTB dysfunction to associate with activation of the p38 mitogen-activated protein kinase pathway. Herein, we discuss the signaling pathways and mechanisms involved in blood-testis barrier restructuring in the mammalian testis.
Assuntos
Barreira Hematotesticular/fisiologia , Células de Sertoli/metabolismo , Testículo/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Comunicação Autócrina , Barreira Hematotesticular/metabolismo , Humanos , Masculino , Comunicação Parácrina , Células de Sertoli/citologia , Transdução de Sinais , Testosterona/metabolismo , Testosterona/fisiologiaRESUMO
Focal adhesion kinase (FAK), as its name implied, is an important mediator of integrin-based signaling function in mammalian cells at the focal adhesion complex (FAC, also known as focal contact) at the cell-extracellular matrix interface. FAK is intimately related to cell movement, such as in macrophages, fibroblasts and also tumor cells. In the testis, however, FAK and two of its phosphorylated forms, p-FAK-Tyr(407) and -Tyr(397), are not found at the FAC since there is no ultrastructure analogous or similar to FAC in the mammalian testis vs. other epithelia. Instead, FAK and its two phosphorylated forms are detected along the seminiferous epithelium in the rat testis at the cell-cell interface in a testis-specific adherens junction (AJ) known as the ectoplasmic specialization (ES). ES is an F-actin-rich ultrastructure in which bundles of actin filaments are sandwiched in-between plasma membrane and cisternae of endoplasmic reticulum not found in other mammalian epithelial/endothelial cells. The ES is restricted to the interface of Sertoli cells and spermatids (step 8-19) known as the apical ES, and to the Sertoli cell-cell interface known as the basal ES. Interestingly, the basal ES is also an integrated component of the blood-testis barrier (BTB), coexisting with tight junction (TJ) and gap junction (GJ), and it is conceivable that actin filament bundles at the ES undergo extensive organization, converting from their "bundled" to "de-bundled/branching" configuration to facilitate transport of germ cells across the epithelium and at the BTB during the epithelial cycle. A recent report (Lie et al. PNAS 109:12562-12567, 2012) has demonstrated that the stage-specific and spatiotemporal expression of p-FAK-Tyr(407) and -Tyr(397) are crucial to the regulation of these events via their stage-specific and spatiotemporal expression during the epithelial cycle mediated by their effects on the organization of the actin filament bundles at the ES, involving actin binding/regulatory proteins. In this Commentary, we will critically evaluate these findings in light of other recent reports in the field. While these ideas are based on studies in the BTB in the rat testis, this information should be applicable and helpful to investigators studying other tissue barriers.
RESUMO
In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas do Citoesqueleto/fisiologia , Fosfoproteínas/fisiologia , Testículo/metabolismo , Testículo/fisiologia , Junções Íntimas/metabolismo , Citoesqueleto de Actina/genética , Fatores Etários , Animais , Barreira Hematotesticular/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Hidrazinas/farmacologia , Indazóis/farmacologia , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Células de Sertoli/fisiologia , Células de Sertoli/ultraestrutura , Testículo/efeitos dos fármacos , Testículo/ultraestrutura , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/genética , Junções Íntimas/fisiologiaRESUMO
Cellular events that occur across the seminiferous epithelium in the mammalian testis during spermatogenesis are tightly coordinated by biologically active peptides released from laminin chains. Laminin-γ3 domain IV is released at the apical ectoplasmic specialization during spermiation and mediates restructuring of the blood-testis barrier, which facilitates the transit of preleptotene spermatocytes. Here we determine the biologically active domain in laminin-γ3 domain IV, which we designate F5 peptide, and show that the overexpression of this domain, or the use of a synthetic F5 peptide, in Sertoli cells with an established functional blood-testis barrier reversibly perturbs blood-testis barrier integrity in vitro and in the rat testis in vivo. This effect is mediated via changes in protein distribution at the Sertoli and Sertoli-germ-cell cell interface and by phosphorylation of focal adhesion kinase at Tyr(407). The consequences are perturbed organization of actin filaments in Sertoli cells, disruption of the blood-testis barrier and spermatid loss. The impairment of spermatogenesis suggests that this laminin peptide fragment may serve as a contraceptive in male rats.
Assuntos
Laminina/química , Peptídeos/farmacologia , Espermatogênese/efeitos dos fármacos , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Barreira Hematotesticular/citologia , Barreira Hematotesticular/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Adesão Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Masculino , Dados de Sequência Molecular , Mutação/genética , Ocludina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Junções Íntimas/metabolismoRESUMO
The blood-testis barrier (BTB) is an important ultrastructure in the testis. A delay in its assembly during postnatal development leads to meiotic arrest. Also, a disruption of the BTB by toxicants in adult rats leads to a failure in spermatogonial differentiation. However, the regulation of BTB assembly remains unknown. Herein, filamin A, an actin filament cross-linker that is known to maintain and regulate cytoskeleton structure and function in other epithelia, was shown to be highly expressed during the assembly of Sertoli cell BTB in vitro and postnatal development of BTB in vivo, perhaps being used to maintain the actin filament network at the BTB. A knockdown of filamin A by RNA interference was found to partially perturb the Sertoli cell tight junction (TJ) permeability barrier both in vitro and in vivo. Interestingly, this down-regulating effect on the TJ barrier function after the knockdown of filamin A was associated with a mis-localization of both TJ and basal ectoplasmic specialization proteins. Filamin A knockdown also induced a disorganization of the actin filament network in Sertoli cells in vitro and in vivo. Collectively, these findings illustrate that filamin A regulates BTB assembly by recruiting these proteins to the microenvironment in the seminiferous epithelium to serve as the building blocks. In short, filamin A participates in BTB assembly by regulating protein recruitment during postnatal development in the rat testis.