ABSTRACT
Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
Subject(s)
Chromaffin Cells , Dynamin II , Endocytosis , Exocytosis , Mutation , Myopathies, Structural, Congenital , Chromaffin Cells/metabolism , Endocytosis/physiology , Endocytosis/genetics , Dynamin II/genetics , Dynamin II/metabolism , Animals , Exocytosis/physiology , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/pathology , Myopathies, Structural, Congenital/metabolism , Mutation/genetics , Cattle , Humans , Actins/metabolism , Actins/genetics , Cells, Cultured , Patch-Clamp Techniques , Adrenal Glands/metabolism , Adrenal Glands/pathologyABSTRACT
IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.
ABSTRACT
Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. Here, we investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. We found that reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, we evaluated the requirement of Prpk in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response.
ABSTRACT
Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.
Subject(s)
Neutrophils , Sepsis , Animals , Disease Models, Animal , Lung/metabolism , Mice , Mice, Inbred C57BL , Neutrophil Infiltration/physiology , Neutrophils/metabolismABSTRACT
Abstract Plants adjust their shoot growth to acclimate to changing environmental factors, such as to enhanced Ultraviolet-B (UV-B) radiation. However, people have ignored that plant roots can also respond to UV-B light. Here, we find the morphology curled wheat roots under UV-B radiation, that we call, "bending roots." The curly region is the transition zone of the root after observed at the cellular level. After exposed to enhanced UV-B radiation for 2 d (10.08 KJ/m2/d), cell size decreased and actin filaments gathered in wheat roots. We also find that H2O2 production increased and that content of the indole-3-acetic acid (IAA) increased remarkably. The pharmacological experiment revealed that actin filaments gathered and polymerized into bundles in the wheat root cells after irrigated H2O2 and IAA. These results indicated that actin filaments changed their distribution and formed the "bending root," which was related to H2O2 production and increase in IAA. Overall, actin filaments in wheat root cells could be a subcellular target of UV-B radiation, and its disruption determines root morphology.
Resumo As plantas ajustam o crescimento da parte aérea para se adaptarem a fatores ambientais variáveis, como o aumento da radiação ultravioleta B (UVB). No entanto, as pessoas ignoram que as raízes das plantas também podem responder à luz UVB. Neste estudo, verificamos a morfologia das raízes enroladas de trigo sob radiação UVB, o que chamamos de "raízes dobradas". A região encaracolada é a zona de transição da raiz no nível celular. Depois de exposição à radiação UVB aprimorada por 2 dias (10,08 KJ/m2/d), o tamanho das células diminuiu, e os filamentos de actina se reuniram. Também constatamos que a produção de H2O2 aumentou e que o conteúdo do ácido indol-3-acético (IAA) aumentou notavelmente. O experimento farmacológico revelou que os filamentos de actina se reuniram e polimerizaram em feixes nas células da raiz de trigo após irrigação com H2O2 e IAA. Esses resultados indicam que os filamentos de actina alteraram sua distribuição e formaram a "raiz dobrada", relacionada à produção de H2O2 e ao aumento do IAA. No geral, os filamentos de actina nas células da raiz de trigo podem ser um alvo subcelular da radiação UVB, e sua interrupção determina a morfologia da raiz.
Subject(s)
Triticum , Hydrogen Peroxide , Ultraviolet Rays , Actin Cytoskeleton , Plant RootsABSTRACT
Plants adjust their shoot growth to acclimate to changing environmental factors, such as to enhanced Ultraviolet-B (UV-B) radiation. However, people have ignored that plant roots can also respond to UV-B light. Here, we find the morphology curled wheat roots under UV-B radiation, that we call, bending roots. The curly region is the transition zone of the root after observed at the cellular level. After exposed to enhanced UV-B radiation for 2 d (10.08 KJ/m2/d), cell size decreased and actin filaments gathered in wheat roots. We also find that H2O2 production increased and that content of the indole-3-acetic acid (IAA) increased remarkably. The pharmacological experiment revealed that actin filaments gathered and polymerized into bundles in the wheat root cells after irrigated H2O2 and IAA. These results indicated that actin filaments changed their distribution and formed the bending root, which was related to H2O2 production and increase in IAA. Overall, actin filaments in wheat root cells could be a subcellular target of UV-B radiation, and its disruption determines root morphology.(AU)
As plantas ajustam o crescimento da parte aérea para se adaptarem a fatores ambientais variáveis, como o aumento da radiação ultravioleta B (UVB). No entanto, as pessoas ignoram que as raízes das plantas também podem responder à luz UVB. Neste estudo, verificamos a morfologia das raízes enroladas de trigo sob radiação UVB, o que chamamos de raízes dobradas. A região encaracolada é a zona de transição da raiz no nível celular. Depois de exposição à radiação UVB aprimorada por 2 dias (10,08 KJ/m2/d), o tamanho das células diminuiu, e os filamentos de actina se reuniram. Também constatamos que a produção de H2O2 aumentou e que o conteúdo do ácido indol-3-acético (IAA) aumentou notavelmente. O experimento farmacológico revelou que os filamentos de actina se reuniram e polimerizaram em feixes nas células da raiz de trigo após irrigação com H2O2 e IAA. Esses resultados indicam que os filamentos de actina alteraram sua distribuição e formaram a raiz dobrada, relacionada à produção de H2O2 e ao aumento do IAA. No geral, os filamentos de actina nas células da raiz de trigo podem ser um alvo subcelular da radiação UVB, e sua interrupção determina a morfologia da raiz.(AU)
Subject(s)
Triticum/radiation effects , Plant Roots/radiation effects , Actins , Ultraviolet Rays/adverse effectsABSTRACT
BACKGROUND: UV-B signaling in plants is mediated by UVR8, which interacts with transcriptional factors to induce root morphogenesis. However, research on the downstream molecules of UVR8 signaling in roots is still scarce. As a wide range of functional cytoskeletons, how actin filaments respond to UV-B-induced root morphogenesis has not been reported. The aim of this study was to investigate the effect of actin filaments on root morphogenesis under UV-B and hydrogen peroxide exposure in Arabidopsis. RESULTS: A Lifeact-Venus fusion protein was used to stain actin filaments in Arabidopsis. The results showed that UV-B inhibited hypocotyl and root elongation and caused an increase in H2O2 content only in the root but not in the hypocotyl. Additionally, the actin filaments in hypocotyls diffused under UV-B exposure but were gathered in a bundle under the control conditions in either Lifeact-Venus or uvr8 plants. Exogenous H2O2 inhibited root elongation in a dose-dependent manner. The actin filaments changed their distribution from filamentous to punctate in the root tips and mature regions at a lower concentration of H2O2 but aggregated into thick bundles with an abnormal orientation at H2O2 concentrations up to 2 mM. In the root elongation zone, the actin filament arrangement changed from lateral to longitudinal after exposure to H2O2. Actin filaments in the root tip and elongation zone were depolymerized into puncta under UV-B exposure, which showed the same tendency as the low-concentration treatments. The actin filaments were hardly filamentous in the maturation zone. The dynamics of actin filaments in the uvr8 group under UV-B exposure were close to those of the control group. CONCLUSIONS: The results indicate that UV-B inhibited Arabidopsis hypocotyl elongation by reorganizing actin filaments from bundles to a loose arrangement, which was not related to H2O2. UV-B disrupted the dynamics of actin filaments by changing the H2O2 level in Arabidopsis roots. All these results provide an experimental basis for investigating the interaction of UV-B signaling with the cytoskeleton.
Subject(s)
Actin Cytoskeleton/physiology , Arabidopsis/growth & development , Hydrogen Peroxide/pharmacology , Plant Roots/growth & development , Ultraviolet Rays , Arabidopsis/radiation effects , Arabidopsis Proteins , Chromosomal Proteins, Non-HistoneABSTRACT
BACKGROUND: UV-B signaling in plants is mediated by UVR8, which interacts with transcriptional factors to induce root morphogenesis. However, research on the downstream molecules of UVR8 signaling in roots is still scarce. As a wide range of functional cytoskeletons, how actin filaments respond to UV-B-induced root morphogenesis has not been reported. The aim of this study was to investigate the effect of actin filaments on root morphogenesis under UV-B and hydrogen peroxide exposure in Arabidopsis. RESULTS: A Lifeact-Venus fusion protein was used to stain actin filaments in Arabidopsis. The results showed that UV-B inhibited hypocotyl and root elongation and caused an increase in H2O2 content only in the root but not in the hypocotyl. Additionally, the actin filaments in hypocotyls diffused under UV-B exposure but were gathered in a bundle under the control conditions in either Lifeact-Venus or uvr8 plants. Exogenous H2O2 inhibited root elongation in a dose-dependent manner. The actin filaments changed their distribution from filamentous to punctate in the root tips and mature regions at a lower concentration of H2O2 but aggregated into thick bundles with an abnormal orientation at H2O2 concentrations up to 2 mM. In the root elongation zone, the actin filament arrangement changed from lateral to longitudinal after exposure to H2O2. Actin filaments in the root tip and elongation zone were depolymerized into puncta under UV-B exposure, which showed the same tendency as the low-concentration treatments. The actin filaments were hardly filamentous in the maturation zone. The dynamics of actin filaments in the uvr8 group under UV-B exposure were close to those of the control group. CONCLUSIONS: The results indicate that UV-B inhibited Arabidopsis hypocotyl elongation by reorganizing actin filaments from bundles to a loose arrangement, which was not related to H2O2. UV-B disrupted the dynamics of actin filaments by changing the H2O2 level in Arabidopsis roots. All these results provide an experimental basis for investigating the interaction of UV-B signaling with the cytoskeleton.
Subject(s)
Ultraviolet Rays , Actin Cytoskeleton/physiology , Arabidopsis/growth & development , Plant Roots/growth & development , Hydrogen Peroxide/pharmacology , Chromosomal Proteins, Non-Histone , Arabidopsis/radiation effects , Arabidopsis ProteinsABSTRACT
Abstract Plants adjust their shoot growth to acclimate to changing environmental factors, such as to enhanced Ultraviolet-B (UV-B) radiation. However, people have ignored that plant roots can also respond to UV-B light. Here, we find the morphology curled wheat roots under UV-B radiation, that we call, bending roots. The curly region is the transition zone of the root after observed at the cellular level. After exposed to enhanced UV-B radiation for 2 d (10.08 KJ/m2/d), cell size decreased and actin filaments gathered in wheat roots. We also find that H2O2 production increased and that content of the indole-3-acetic acid (IAA) increased remarkably. The pharmacological experiment revealed that actin filaments gathered and polymerized into bundles in the wheat root cells after irrigated H2O2 and IAA. These results indicated that actin filaments changed their distribution and formed the bending root, which was related to H2O2 production and increase in IAA. Overall, actin filaments in wheat root cells could be a subcellular target of UV-B radiation, and its disruption determines root morphology.
Resumo As plantas ajustam o crescimento da parte aérea para se adaptarem a fatores ambientais variáveis, como o aumento da radiação ultravioleta B (UVB). No entanto, as pessoas ignoram que as raízes das plantas também podem responder à luz UVB. Neste estudo, verificamos a morfologia das raízes enroladas de trigo sob radiação UVB, o que chamamos de raízes dobradas. A região encaracolada é a zona de transição da raiz no nível celular. Depois de exposição à radiação UVB aprimorada por 2 dias (10,08 KJ/m2/d), o tamanho das células diminuiu, e os filamentos de actina se reuniram. Também constatamos que a produção de H2O2 aumentou e que o conteúdo do ácido indol-3-acético (IAA) aumentou notavelmente. O experimento farmacológico revelou que os filamentos de actina se reuniram e polimerizaram em feixes nas células da raiz de trigo após irrigação com H2O2 e IAA. Esses resultados indicam que os filamentos de actina alteraram sua distribuição e formaram a raiz dobrada, relacionada à produção de H2O2 e ao aumento do IAA. No geral, os filamentos de actina nas células da raiz de trigo podem ser um alvo subcelular da radiação UVB, e sua interrupção determina a morfologia da raiz.
ABSTRACT
ß-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.
Subject(s)
Actins/metabolism , Calcium Channels, L-Type/metabolism , Chromaffin Cells/metabolism , Down-Regulation/physiology , src Homology Domains/physiology , Animals , Cattle , Cells, Cultured , Protein Subunits/metabolism , Protein Transport/physiology , RabbitsABSTRACT
Several studies have shown that a single exposure to stress may improve or impair learning and memory processes, depending on the timing in which the stress event occurs with relation to the acquisition phase. However, to date there is no information about the molecular changes that occur at the synapse during the stress-induced memory modification and after a recovery period. In particular, there are no studies that have evaluated-at the same time-the temporality of stress and stress recovery period in hippocampal short-term memory and the effects on dendritic spine morphology, along with variations in N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits. The aim of our study was to take a multidimensional approach to investigate concomitant behavioral, morphological and molecular changes induced by a single restraint stress exposure (2.5 h) and a recovery period of 6 and 24 h in rats. We found that acute stress elicited a reduced preference to explore an object placed in a novel position (a hippocampal-dependent task). These changes were accompanied by increased activity of LIM kinase I (LIMK; an actin-remodeling protein) and increased levels of NR2A subunits of NMDA receptors. After 6 h of recovery from stress, rats showed similar preference to explore an object placed in a novel or familiar position, but density of immature spines increased in secondary CA1 apical dendrites, along with a transient rise in GluA2 AMPA receptor subunits. After 24 h of recovery from stress, the animals showed a preference to explore an object placed in a novel position, which was accompanied by a normalization of NMDA and AMPA receptor subunits to control values. Our data suggest that acute stress produces reversible molecular and behavioral changes 24 h after stress, allowing a full reestablishment of hippocampal-related memory. Further studies need to be conducted to deepen our understanding of these changes and their reciprocal interactions.Adaptive stress responses are a promising avenue to develop interventions aiming at restoring hippocampal function impaired by repetitive stress exposure.
ABSTRACT
Abstract Plants adjust their shoot growth to acclimate to changing environmental factors, such as to enhanced Ultraviolet-B (UV-B) radiation. However, people have ignored that plant roots can also respond to UV-B light. Here, we find the morphology curled wheat roots under UV-B radiation, that we call, bending roots. The curly region is the transition zone of the root after observed at the cellular level. After exposed to enhanced UV-B radiation for 2 d (10.08 KJ/m2/d), cell size decreased and actin filaments gathered in wheat roots. We also find that H2O2 production increased and that content of the indole-3-acetic acid (IAA) increased remarkably. The pharmacological experiment revealed that actin filaments gathered and polymerized into bundles in the wheat root cells after irrigated H2O2 and IAA. These results indicated that actin filaments changed their distribution and formed the bending root, which was related to H2O2 production and increase in IAA. Overall, actin filaments in wheat root cells could be a subcellular target of UV-B radiation, and its disruption determines root morphology.
Resumo As plantas ajustam o crescimento da parte aérea para se adaptarem a fatores ambientais variáveis, como o aumento da radiação ultravioleta B (UVB). No entanto, as pessoas ignoram que as raízes das plantas também podem responder à luz UVB. Neste estudo, verificamos a morfologia das raízes enroladas de trigo sob radiação UVB, o que chamamos de raízes dobradas. A região encaracolada é a zona de transição da raiz no nível celular. Depois de exposição à radiação UVB aprimorada por 2 dias (10,08 KJ/m2/d), o tamanho das células diminuiu, e os filamentos de actina se reuniram. Também constatamos que a produção de H2O2 aumentou e que o conteúdo do ácido indol-3-acético (IAA) aumentou notavelmente. O experimento farmacológico revelou que os filamentos de actina se reuniram e polimerizaram em feixes nas células da raiz de trigo após irrigação com H2O2 e IAA. Esses resultados indicam que os filamentos de actina alteraram sua distribuição e formaram a raiz dobrada, relacionada à produção de H2O2 e ao aumento do IAA. No geral, os filamentos de actina nas células da raiz de trigo podem ser um alvo subcelular da radiação UVB, e sua interrupção determina a morfologia da raiz.