Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

AbstractKeratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

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Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

10.1101/868034 ◽

2019 ◽

Author(s):

Clare Rogerson ◽

Duncan Wotherspoon ◽

Ryan F L O’Shaughnessy

Keyword(s):

Actin Filament ◽

Molecular Mechanisms ◽

Molecular Motor ◽

Nuclear Lamina ◽

Nuclear Volume ◽

Volume Reduction ◽

Epidermal Keratinocytes ◽

Lamin A ◽

Myosin Iia

AbstractKeratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 minutes precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. Single cell RNA sequencing of differentiating keratinocytes identified gene changes correlated with lamin dispersal, which we propose are due to changes in lamina-associated domains upon Lamin A/C dispersal. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

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Reduced Lamin A/C does Not Facilitate Cancer Cell Transendothelial Migration but Compromises Lung Metastasis

Cancers ◽

10.3390/cancers13102383 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2383

Author(s):

Francesco Roncato ◽

Ofer Regev ◽

Sara W. Feigelson ◽

Sandeep Kumar Yadav ◽

Lukasz Kaczmarczyk ◽

...

Keyword(s):

Cancer Cells ◽

Lung Metastasis ◽

Metastatic Cancer ◽

Nuclear Lamina ◽

Transendothelial Migration ◽

Soft Agar ◽

Lamin A ◽

And Migration

The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.

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Abstract MP175: Disruption of the Genome Architecture at the PRRX1 Locus is Associated With the Pathogenesis of LMNA -related Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.127.suppl_1.mp175 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Yuan Zhang ◽

Mohamed Ameen ◽

Isaac Perea Gil ◽

Jennifer Arthur ◽

Alexandra A Gavidia ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Single Cell ◽

Genome Organization ◽

Molecular Mechanisms ◽

Critical Role ◽

Lamin A ◽

Candidate Locus ◽

Gene Encoding ◽

Gene Dysregulation

Background: LMNA , a gene encoding A-type lamin proteins (abbreviated as lamin A), is one of the most frequently mutated genes in dilated cardiomyopathy (DCM). The molecular mechanisms underlying cardiomyocyte dysfunction in LMNA -related DCM remain elusive, translating to the lack of disease-specific therapies. Lamin A has been shown to play a critical role in genome organization via interactions with the chromatin at specific regions called lamina-associated domains (LADs). However, little is known about whether DCM-causing LMNA mutations rearrange the genome conformation and chromosome accessibility. The overarching goal of this study is to define the role of genome organization in LMNA -related DCM. Methods: LMNA -related DCM was modeled in vitro using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from DCM patients carrying a frameshift mutation in the LMNA gene (c. 348_349insG; p. K117fs) and isogenic controls. We combined genome-wide single cell functional genomic and epigenomic mapping analyses to define the gene regulation and cis-regulatory interactions in isogenic iPSC-CMs. Results: Single-cell RNA-seq revealed global gene dysregulation in LMNA mutant compared to isogenic control iPSC-CMs. The homeodomain transcription factor PRRX1 was significantly upregulated in mutant cells. We showed that LAD integrity is disrupted at the PRRX1 locus in mutant iPSC-CMs. In agreement, DNA fluorescence in situ hybridization (FISH) revealed that the PRRX1 locus loses peripheral association and relocates towards the transcriptionally active nuclear interior in mutant iPSC-CMs. Correspondingly, single-cell assay for transposase accessible chromatin (ATAC)-seq showed increased chromatin co-accessibility at the PRRX1 locus, providing a plausible explanation for ectopic activation of PRRX1 in LMNA mutant iPSC-CMs. Conclusion: Our data suggest that LMNA haploinsufficiency disrupts the structure of LADs, leading to ectopic promoter interactions and altered gene expression in LMNA -related DCM iPSC-CMs. We identified PRRX1 as a promising candidate locus linking changes in LAD organization with gene dysregulation in LMNA -related DCM.

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Cellular and Animal Models of Striated Muscle Laminopathies

Cells ◽

10.3390/cells8040291 ◽

2019 ◽

Vol 8 (4) ◽

pp. 291 ◽

Cited By ~ 3

Author(s):

Hannah A. Nicolas ◽

Marie-Andrée Akimenko ◽

Frédérique Tesson

Keyword(s):

Skeletal Muscle ◽

Skeletal Muscles ◽

Molecular Mechanisms ◽

Striated Muscle ◽

Nuclear Lamina ◽

The Other ◽

Lamin A ◽

Lmna Gene ◽

Future Directions ◽

Exon 10

The lamin A/C (LMNA) gene codes for nuclear intermediate filaments constitutive of the nuclear lamina. LMNA has 12 exons and alternative splicing of exon 10 results in two major isoforms—lamins A and C. Mutations found throughout the LMNA gene cause a group of diseases collectively known as laminopathies, of which the type, diversity, penetrance and severity of phenotypes can vary from one individual to the other, even between individuals carrying the same mutation. The majority of the laminopathies affect cardiac and/or skeletal muscles. The underlying molecular mechanisms contributing to such tissue-specific phenotypes caused by mutations in a ubiquitously expressed gene are not yet well elucidated. This review will explore the different phenotypes observed in established models of striated muscle laminopathies and their respective contributions to advancing our understanding of cardiac and skeletal muscle-related laminopathies. Potential future directions for developing effective treatments for patients with lamin A/C mutation-associated cardiac and/or skeletal muscle conditions will be discussed.

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The dynamic instability of actin filament barbed ends

The Journal of Cell Biology ◽

10.1083/jcb.202102020 ◽

2021 ◽

Vol 220 (4) ◽

Author(s):

Guillaume Romet-Lemonne ◽

Antoine Jégou

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Molecular Mechanisms ◽

Dynamic Instability ◽

Filament Networks ◽

In Cells

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

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Local Actin Filament Geometry Dictates How Myosin Va Molecular Motor Teams Transport Liposomes Through 3D Actin Networks in Vitro

Biophysical Journal ◽

10.1016/j.bpj.2018.11.697 ◽

2019 ◽

Vol 116 (3) ◽

pp. 125a

Author(s):

Sam Walcott ◽

Andrew T. Lombardo ◽

Kathleen M. Trybus ◽

David M. Warshaw

Keyword(s):

Actin Filament ◽

Molecular Motor ◽

Actin Networks ◽

Myosin Va

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Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

Life Science Alliance ◽

10.26508/lsa.202101120 ◽

2021 ◽

Vol 4 (10) ◽

pp. e202101120

Author(s):

Ching-Tung Chu ◽

Yi-Hsuan Chen ◽

Wen-Tai Chiu ◽

Hong-Chen Chen

Keyword(s):

Tyrosine Phosphorylation ◽

Genomic Instability ◽

Posttranslational Modifications ◽

Nuclear Lamina ◽

Serine Phosphorylation ◽

Lamin A ◽

Intact Cells ◽

Structure And Activity

Lamins form the nuclear lamina, which is important for nuclear structure and activity. Although posttranslational modifications, in particular serine phosphorylation, have been shown to be important for structural properties and functions of lamins, little is known about the role of tyrosine phosphorylation in this regard. In this study, we found that the constitutively active Src Y527F mutant caused the disassembly of lamin A/C. We demonstrate that Src directly phosphorylates lamin A mainly at Tyr45 both in vitro and in intact cells. The phosphomimetic Y45D mutant was diffusively distributed in the nucleoplasm and failed to assemble into the nuclear lamina. Depletion of lamin A/C in HeLa cells induced nuclear dysmorphia and genomic instability as well as increased nuclear plasticity for cell migration, all of which were partially restored by re-expression of lamin A, but further promoted by the Y45D mutant. Together, our results reveal a novel mechanism for regulating the assembly of nuclear lamina through Src and suggest that aberrant phosphorylation of lamin A by Src may contribute to nuclear dysmorphia, genomic instability, and nuclear plasticity.

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Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.117 ◽

1987 ◽

Vol 105 (1) ◽

pp. 117-125 ◽

Cited By ~ 199

Author(s):

S D Georgatos ◽

G Blobel

Keyword(s):

Plasma Membrane ◽

Specific Binding ◽

Attachment Site ◽

Nuclear Lamina ◽

Membrane Skeleton ◽

Eukaryotic Cells ◽

Lamin A ◽

Lamin B ◽

Nuclear Lamins

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

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Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear Lamina To Facilitate Virion Production

Journal of Virology ◽

10.1128/jvi.01100-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11913-11926 ◽

Cited By ~ 75

Author(s):

Chung-Pei Lee ◽

Yu-Hao Huang ◽

Su-Fang Lin ◽

Yao Chang ◽

Yu-Hsin Chang ◽

...

Keyword(s):

Epstein Barr Virus ◽

Fluorescent Protein ◽

Nuclear Lamina ◽

Lamin A ◽

Genome Replication ◽

Barr Virus ◽

Virion Production ◽

Cellular Environment ◽

Epstein Barr

ABSTRACT DNA viruses adopt various strategies to modulate the cellular environment for efficient genome replication and virion production. Previously, we demonstrated that the BGLF4 kinase of Epstein-Barr virus (EBV) induces premature chromosome condensation through the activation of condensin and topoisomerase IIα (C. P. Lee, J. Y. Chen, J. T. Wang, K. Kimura, A. Takemoto, C. C. Lu, and M. R. Chen, J. Virol. 81:5166-5180, 2007). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha- and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.

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Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Journal of Virology ◽

10.1128/jvi.02426-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 523-534 ◽

Cited By ~ 35

Author(s):

Mayuri Sharma ◽

Brian J. Bender ◽

Jeremy P. Kamil ◽

Ming F. Lye ◽

Jean M. Pesola ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Continuous Distribution ◽

Virus Production ◽

Nuclear Lamina ◽

Dominant Negative ◽

Lamin A ◽

Nuclear Egress ◽

Dividing Cells ◽

Infected Cells

ABSTRACTHerpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with aUL97mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

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