Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

Schweinhuber, Stefanie Katharina; Meßerschmidt, Tania; Hänsch, Robert; Körte, Martin; Rothkegel, Martin

doi:10.1371/journal.pone.0117244

Cited by 16 publications

(19 citation statements)

References 69 publications

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“…We think this is unlikely since phalloidin staining did not reveal any F-actin clusters at the sites of T89D-Pfn1 aggregates. Although it remains to be seen whether activating the PKA pathway triggers T89 phosphorylation of Pfn1 in cells, if T89-phosphorylation of Pfn1 inhibits actin polymerization of Pfn1 as suggested by the F-actin phenotype of T89D-Pfn1 expressors in this study, it would be at least consistent with the recent finding of FSK-induced stellation of astrocytes in a Pfn-dependent manner [ 18 ].…”

Section: Discussionsupporting

confidence: 90%

“…PKC-mediated phosphorylation of Pfn1 on S137 residue has been characterized previously [ 11 , 12 ]. Based on alterations in post-translational modification patterns of Pfn isoforms in astrocytes upon treatment with FSK (an activator of cyclic AMP-PKA pathway), it has been recently suggested that PKA may be involved in post-translational regulation of Pfn isoforms [ 18 ]. Whether Pfn1 is a phosphorylation substrate of PKA however remains unknown.…”

Section: Resultsmentioning

confidence: 99%

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Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

et al. 2016

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ObjectiveDynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells.MethodologyWe performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1.Results and SignificanceWe identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes.

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Section: Discussionsupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

et al. 2016

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“…Src and FAK both mediate signaling pathways that regulate actin polymerization [ 49 , 54 ]. Consistent with this idea, both protein and phosphorylation levels of profilin, an actin-binding protein that regulates actin polymerization and astrocytic morphology [ 55 ], was increased in the cortices of Mlc1 -ErbB2 V664E mice ( Figure 7a and b ).…”

Section: Resultssupporting

confidence: 61%

A role for ErbB signaling in the induction of reactive astrogliosis

Chen

et al. 2017

Cell Discov

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Reactive astrogliosis is a hallmark of many neurological disorders, yet its functions and molecular mechanisms remain elusive. Particularly, the upstream signaling that regulates pathological responses of astrocytes is largely undetermined. We used a mouse traumatic brain injury model to induce astrogliosis and revealed activation of ErbB receptors in reactive astrocytes. Moreover, cell-autonomous inhibition of ErbB receptor activity in reactive astrocytes by a genetic approach suppressed hypertrophic remodeling possibly through the regulation of actin dynamics. However, inhibiting ErbB signaling in reactive astrocytes did not affect astrocyte proliferation after brain injury, although it aggravated local inflammation. In contrast, active ErbB signaling in mature astrocytes of various brain regions in mice was sufficient to initiate reactive responses, reproducing characterized molecular and cellular features of astrogliosis observed in injured or diseased brains. Further, prevalent astrogliosis in the brain induced by astrocytic ErbB activation caused anorexia in animals. Therefore, our findings defined an unrecognized role of ErbB signaling in inducing reactive astrogliosis. Mechanistically, inhibiting ErbB signaling in reactive astrocytes prominently reduced Src and focal adhesion kinase (FAK) activity that is important for actin remodeling, although ErbB signaling activated multiple downstream signaling proteins. The discrepancies between the results from loss- and gain-of-function studies indicated that ErbB signaling regulated hypertrophy and proliferation of reactive astrocytes by different downstream signaling pathways. Our work demonstrated an essential mechanism in the pathological regulation of astrocytes and provided novel insights into potential therapeutic targets for astrogliosis-implicated diseases.

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“…Isoform-specific knockdowns of either ubiquitous profilin 1 or CNS-specific profilin 2a which is involved in neurons in presynaptic membrane trafficking and dendritic spine remodeling (Pilo Boyl et al, 2007 ; Michaelsen et al, 2010 ), reduce the total volume of astrocytes in organotypic slices. However, selective inhibition of profilin 1 affects the number and movement of filopodia processes by slowing the reorganization of filamentous actin (Molotkov et al, 2013 ; Schweinhuber et al, 2015 ).…”

Section: A Starring Role For Astrocytes At Synapsesmentioning

confidence: 99%

Important Shapeshifter: Mechanisms Allowing Astrocytes to Respond to the Changing Nervous System During Development, Injury and Disease

Schiweck

Eickholt

Murk

2018

Front. Cell. Neurosci.

177

188

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Astrocytes are the most prevalent glial cells in the brain. Historically considered as “merely supporting” neurons, recent research has shown that astrocytes actively participate in a large variety of central nervous system (CNS) functions including synaptogenesis, neuronal transmission and synaptic plasticity. During disease and injury, astrocytes efficiently protect neurons by various means, notably by sealing them off from neurotoxic factors and repairing the blood-brain barrier. Their ramified morphology allows them to perform diverse tasks by interacting with synapses, blood vessels and other glial cells. In this review article, we provide an overview of how astrocytes acquire their complex morphology during development. We then move from the developing to the mature brain, and review current research on perisynaptic astrocytic processes, with a particular focus on how astrocytes engage synapses and modulate their formation and activity. Comprehensive changes have been reported in astrocyte cell shape in many CNS pathologies. Factors influencing these morphological changes are summarized in the context of brain pathologies, such as traumatic injury and degenerative conditions. We provide insight into the molecular, cellular and cytoskeletal machinery behind these shape changes which drive the dynamic remodeling in astrocyte morphology during injury and the development of pathologies.

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Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

Cited by 16 publications

References 69 publications

Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

A role for ErbB signaling in the induction of reactive astrogliosis

Important Shapeshifter: Mechanisms Allowing Astrocytes to Respond to the Changing Nervous System During Development, Injury and Disease

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