8 research outputs found
Characterisation of phospholipase C-Ī· enzymes and their relevance to disease
Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane
phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)Pā) into the second messengers,
inositol trisphosphate (Ins(4,5)Pā) and diacylglycerol (DAG). Six classes of PLC enzymes have
been identified based on their structure and mechanism of activation.
PLCĪ·s are the most recently identified family and consist of two isozymes, PLCĪ·1 and PLCĪ·2.
The aim of this thesis is to further understand the mechanisms of PLCĪ· activation, the role of
PLCĪ·2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were
found to be activated by physiological concentrations of intracellular CaĀ²āŗ. Activation of
PLCĪ·2 by GĆāĪ³ā was confirmed using a bacterial 2A co-expression system to allow expression
of PLCĪ·2, GĆā and GĪ³ā with a single plasmid. Localisation studies show a nuclear distribution
for PLCĪ·2, but a cytoplasmic distribution for PLCĪ·1 in a neuroblastoma cells line (Neuro2A).
PLCĪ·2 has been implicated in brain development and neurite formation. Building on this, a
neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms
of PLCĪ·2 was utilised, revealing that PLCĪ·2 activity is essential for neuritogenesis but that
this process is independent of the enzymes high sensitivity towards CaĀ²āŗ. Furthermore, the direct interaction of PLCĪ·2 and LIMK-1, a previously identified PLCĪ·2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCĪ· enzymes to CaĀ²āŗ and because of their presence within neurons, they may be involved in CaĀ²āŗ dysregulation that occurs in certain diseases such as Alzheimerās disease (AD). The role of PLCĪ·2 was assessed in amyloid-Ć (AĆ) treated
differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCĪ·1 was investigated due to a recently identified PLCĪ·1 polymorphism in patients with holoprosencephaly, an embryonic midline defect
Phospholipase C-Ī·2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth
We gratefully acknowledge financial support from the Wellcome Trust (Grant No. WT089803MA to J.M.L.) and the School of Medicine, University of St Andrews.Neurite growth is central to the formation and differentiation of functional neurons and recently, an essential role for phospholipase C-Ī·2 (PLCĪ·2) in neuritogenesis was revealed. Here we investigate the function of PLCĪ·2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCĪ·2 calcium-binding EF-hand domain, a domain that is known to be required for calcium-dependent PLCĪ·2 activation. To do this we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCĪ·2 and D256A (EF-hand) and H460Q (active site) PLCĪ·2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCĪ·2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCĪ·2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCĪ·2 by comparing immuno-electron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labelling for PLCĪ·2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCĪ·2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of PLCĪ·2 and its articulation with LIMK-1 in regulating neuritogenesis.Publisher PDFPeer reviewe
Phospholipase C-Ī·2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth
Neurite growth is central to the formation and differentiation of functional neurons and recently, an essential role for phospholipase C-Ī·2 (PLCĪ·2) in neuritogenesis was revealed. Here we investigate the function of PLCĪ·2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCĪ·2 calcium-binding EF-hand domain, a domain that is known to be required for calcium-dependent PLCĪ·2 activation. To do this we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCĪ·2 and D256A (EF-hand) and H460Q (active site) PLCĪ·2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCĪ·2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCĪ·2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCĪ·2 by comparing immuno-electron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labelling for PLCĪ·2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCĪ·2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of PLCĪ·2 and its articulation with LIMK-1 in regulating neuritogenesis
Erratum to: Phospholipase C-Ī·2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth
Phospholipase C-Ī·2 is required for retinoic acid-stimulated neurite growth
PLCĪ·2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCĪ·2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca2+ signals by stimulating further Ca2+ release from Ins(1,4,5)P3-sensitive stores. The role of PLCĪ·2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCĪ·2 expression increased 2-fold during a 4-day differentiation period. Stable expression of PLCĪ·2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA treatment. Furthermore, RA response element (RARE) activation was perturbed by PLCĪ·2 knockdown. Using a bacterial two-hybrid screen we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCĪ·2. Immunostaining of PLCĪ·2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and CREB was reduced in PLCĪ·2 knockdown cells. The phosphoinositide-binding properties of the PLCĪ·2 PH domain, assessed using a FRET-based assay, reveal this domain to possess a high affinity toward PtdIns(3,4,5)P3. Immunostaining of PLCĪ·2 together with PtdIns(3,4,5)P3 in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P3 levels in cellular compartments are likely to be important for the spatial control of PLCĪ·2 signaling
Phospholipase C-eta 2 is required for retinoic acid-stimulated neurite growth
PLCĪ·2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCĪ·2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca2+ signals by stimulating further Ca2+ release from Ins(1,4,5)P3-sensitive stores. The role of PLCĪ·2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCĪ·2 expression increased 2-fold during a 4-day differentiation period. Stable expression of PLCĪ·2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA treatment. Furthermore, RA response element (RARE) activation was perturbed by PLCĪ·2 knockdown. Using a bacterial two-hybrid screen we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCĪ·2. Immunostaining of PLCĪ·2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and CREB was reduced in PLCĪ·2 knockdown cells. The phosphoinositide-binding properties of the PLCĪ·2 PH domain, assessed using a FRET-based assay, reveal this domain to possess a high affinity toward PtdIns(3,4,5)P3. Immunostaining of PLCĪ·2 together with PtdIns(3,4,5)P3 in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P3 levels in cellular compartments are likely to be important for the spatial control of PLCĪ·2 signaling