ABSTRACT
The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.
Subject(s)
Cell Cycle Proteins/metabolism , Centrosome/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , ran GTP-Binding Protein/metabolism , Animals , Centrosome/drug effects , Guanosine Triphosphate/metabolism , Hippocampus/cytology , Mice, Inbred C57BL , Microtubules/drug effects , Models, Biological , Morphogenesis/drug effects , Neurites/drug effects , Neurites/metabolism , Neurons/drug effects , Protein Binding/drug effects , Protein Transport/drug effects , Quinazolines/pharmacology , alpha Karyopherins/metabolism , beta Karyopherins/metabolismABSTRACT
Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.
Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Microtubules/metabolism , Neurites/metabolism , Animals , Hippocampus/cytology , Hippocampus/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Phosphorylation , Protein Binding , Protein TransportABSTRACT
Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 µg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 µg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed.
Subject(s)
Aurintricarboxylic Acid/pharmacology , Cell Adhesion/drug effects , Cell Culture Techniques/methods , Hippocampus/cytology , Hippocampus/drug effects , Neurons/cytology , Neurons/drug effects , Actins/metabolism , Analysis of Variance , Animals , CHO Cells , Cell Line, Tumor , Cell Shape/drug effects , Cricetinae , Cricetulus , Culture Media, Serum-Free , Mice , Mice, Inbred C57BL , Surface Properties , Tretinoin/pharmacology , Tubulin/metabolismABSTRACT
Dissociated hippocampal neuron culture has long been the model system of choice for many neuroscientists. The ability to culture dissociated hippocampal neurons from genetically modified mice provides an invaluable tool for studying many neuronal processes. In this study, we established a novel method to culture dissociated hippocampal neurons from embryonic and neonatal mice. Dissociated neurons were cultured in a microchamber between the glass coverslip and the plastic cell container without the use of glial feeder cells. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures that are difficult to achieve without the use of feeder cells.
Subject(s)
Hippocampus/cytology , Neurobiology/instrumentation , Neurobiology/methods , Neurons/cytology , Animals , Animals, Newborn , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cells, Cultured , Female , Hippocampus/physiology , Male , Mice , Mice, Inbred C57BL , Neurons/physiology , PregnancyABSTRACT
BACKGROUND: Care attendants constitute the main workforce in nursing homes, but their heavy workload, low autonomy, and indefinite responsibility result in high levels of stress and may affect quality of care. However, few studies have focused of this problem. OBJECTIVES: The aim of this study was to examine work-related stress and associated factors that affect care attendants in nursing homes and to offer suggestions for how management can alleviate these problems in care facilities. METHODS: We recruited participants from nine nursing homes with 50 or more beds located in middle Taiwan; 110 care attendants completed the questionnaire. The work stress scale for the care attendants was validated and achieved good reliability (Cronbach's alpha=0.93). We also conducted exploratory factor analysis. RESULTS: Six factors were extracted from the work stress scale: insufficient ability, stressful reactions, heavy workload, trouble in care work, poor management, and working time problems. The explained variance achieved 64.96%. Factors related to higher work stress included working in a hospital-based nursing home, having a fixed schedule, night work, feeling burden, inconvenient facility, less enthusiasm, and self-rated higher stress. CONCLUSION: Work stress for care attendants in nursing homes is related to human resource management and quality of care. We suggest potential management strategies to alleviate work stress for these workers.
