Profilin Isoforms in Health and Disease – All the Same but Different

Murk, Kai; Ornaghi, Marta; Schiweck, Juliane

doi:10.3389/fcell.2021.681122

Cited by 29 publications

(20 citation statements)

References 155 publications

(243 reference statements)

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“…Mutations in PFN1 are associated with neurodegenerative disease, including amyotrophic lateral sclerosis (ALS) (Wu et al, 2012;Murk et al, 2021). Therefore, we generated and confirmed two clonal CRISPR/Cas9 knockout lines (PFN1 (-/-) ) in immortalized Neuroblastoma-2a (N2a) cells (Figure 6A and Figure S7A-C PFN1 deficient cell lines did not proliferate as efficiently as endogenous controls (Figure 6B), consistent with previously reported cell cycle defects (Suetsugu et al, 1998;Witke et al, 2001;Moens and Coumans, 2015).…”

Section: Mapple-pfn1 Restores Endogenous Protein Levels and Cell Func...supporting

confidence: 87%

“…Mutations in PFN1 are associated with neurodegenerative disease, including amyotrophic lateral sclerosis (ALS) (Wu et al, 2012; Murk et al, 2021). Therefore, we generated and confirmed two clonal CRISPR/Cas9 knockout lines (PFN1 (-/-) ) in immortalized Neuroblastoma-2a (N2a) cells (Figure 6A and Figure S7A-C) that have been successfully used to model features of ALS (De Vos et al, 2007; Vance et al, 2009; Coussee et al, 2011; Henty-Ridilla et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

“…Finally, several disease-variants of PFN1 have been identified in cancer and neurodegenerative disorders (Michaelsen-Preusse et al, 2016; Pimm et al, 2020; Murk et al, 2021). Using a genetic approach, we observed less microtubule association in cells expressing the actin-and microtubule-binding deficient PFN1(G118V) ALS variant.…”

Section: Discussionmentioning

confidence: 99%

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Visualizing Molecules of FunctionalHumanProfilin

Pimm

Liu

Tuli

et al. 2021

Preprint

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Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations (121 uM) present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered a fluorescently-labeled profilin that retains native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. This fluorescent profilin directly binds to dimers of tubulin (kD = 1.7 uM) and the microtubule lattice (kD = 10 uM) to stimulate microtubule assembly. In cells, our tagged profilin fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. Thus, this labeled profilin-1 is a reliable tool to investigate the dynamic interactions of profilin with actin or microtubules in live cell and in vitro applications.

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Section: Mapple-pfn1 Restores Endogenous Protein Levels and Cell Func...supporting

confidence: 87%

“…Mutations in PFN1 are associated with neurodegenerative disease, including amyotrophic lateral sclerosis (ALS) (Wu et al, 2012; Murk et al, 2021). Therefore, we generated and confirmed two clonal CRISPR/Cas9 knockout lines (PFN1 (-/-) ) in immortalized Neuroblastoma-2a (N2a) cells (Figure 6A and Figure S7A-C) that have been successfully used to model features of ALS (De Vos et al, 2007; Vance et al, 2009; Coussee et al, 2011; Henty-Ridilla et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

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Visualizing Molecules of FunctionalHumanProfilin

Pimm

Liu

Tuli

et al. 2021

Preprint

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“…Profilin is an essential regulator of the neuronal cytoskeleton through many direct (i.e. binding actin monomers, tubulin dimers, or microtubules) and indirect mechanisms linked to cellular cofactors like formin, Ena/VASP, SMN, and exportin-6 (Figure 6A)(Bowerman et al, 2009; Henty-Ridilla et al, 2017; Murk et al, 2021; Pimm et al, 2021; Skruber et al, 2020; Stüven et al, 2003; Suarez and Kovar, 2016). However, the mechanisms defining the function of the ALS-associated mutations in actin assembly are challenging to interpret from cell-based studies and not well defined or quantitatively compared across all variants.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Liu¹,

Pimm²,

Haarer³

et al. 2022

Preprint

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Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin-mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constants of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-PLP interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

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“…The PFN gene family contains four members, with profilin 1 and 2 displaying close similarity (>60% homology) but PFN3 and PFN4 only sharing <40% and <20% amino acids with other Profilin isoforms [27]. The predicted 3D structures of all isoforms, however, are clearly related (https://alphafold.ebi.ac.uk/, accessed on 10 December 2021).…”

Section: The Slc34a1/pfn3 Locusmentioning

confidence: 99%

Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3)

Zinad

Sae-Lee

Ariza-Mateos

et al. 2022

ncRNA

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Natural antisense transcripts (NATs) constitute a significant group of regulatory, long noncoding RNAs. They are prominently expressed in testis but are also detectable in other organs. NATs are transcribed at low levels and co-expressed with related protein coding sense transcripts. Nowadays NATs are generally considered as regulatory, long noncoding RNAs without closer focus on the inevitable interference between sense and antisense expression. This work describes a cellular system where sense and antisense transcription of a specific locus (SLC34A1/PFN3) is induced using epigenetic modifiers and CRISPR-Cas9. The renal cell lines HEK293 and HKC-8 do not express SLC34A1/PFN3 under normal culture conditions. Five-day exposure to dexamethasone significantly stimulates sense transcript (SLC34A1) levels and antisense (PFN3) minimally; the effect is only seen in HEK293 cells. Enhanced expression is paralleled by reduced sense promoter methylation and an increase in activating histone marks. Expression is further modulated by cassettes that stimulate the expression of sense or antisense transcript but disrupt protein coding potential. Constitutive expression of a 5′-truncated SLC34A1 transcript increases sense expression independent of dexamethasone induction but also stimulates antisense expression. Concordant expression is confirmed with the antisense knock-in that also enhances sense expression. The antisense effect acts on transcription in cis since transient transfection with sense or antisense constructs fails to stimulate the expression of the opposite transcript. These results suggest that bi-directional transcription of the SLC34A1/PFN3 locus has a stimulatory influence on the expression of the opposite transcript involving epigenetic changes of the promoters. In perspective of extensive, previous research into bi-directionally transcribed SLC34A loci, the findings underpin a hypothesis where NATs display different biological roles in soma and germ cells. Accordingly, we propose that in somatic cells, NATs act like lncRNAs–with the benefit of close proximity to a potential target gene. In germ cells, however, recent evidence suggests different biological roles for NATs that require RNA complementarity and double-stranded RNA formation.

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Profilin Isoforms in Health and Disease – All the Same but Different

Cited by 29 publications

References 155 publications

Visualizing Molecules of FunctionalHumanProfilin

Visualizing Molecules of FunctionalHumanProfilin

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3)

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