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Circulation Research Aug 2023FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular...
BACKGROUND
FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood.
METHODS
We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations.
RESULTS
Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components.
CONCLUSIONS
Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.
Topics: Mice; Animals; Filamins; Actins; Muscle, Skeletal; Cardiomyopathies; Myocytes, Cardiac; Mutation; Mammals
PubMed: 37492967
DOI: 10.1161/CIRCRESAHA.123.322750 -
Nucleus (Austin, Tex.) Dec 2024The view on nuclear filaments formed by non-skeletal β-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte... (Review)
Review
The view on nuclear filaments formed by non-skeletal β-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte nuclei and only under specific cell stress conditions in mammalian cell nuclei. Improved labeling and imaging technologies have permitted insights into a transient but microscopically apparent filament network that is relevant for chromatin organization, biomechanics of the mammalian cell nucleus, gene expression, and DNA damage repair. Here, we will provide a historical perspective on the developing insight into nuclear actin filaments.
Topics: Animals; Cell Nucleus; Actin Cytoskeleton; Actins; Cytoskeleton; Oocytes; Mammals
PubMed: 38384139
DOI: 10.1080/19491034.2024.2320656 -
Journal of Biochemistry Feb 2024Blebs are membrane structures formed by the detachment of the plasma membrane from the underlying actin cytoskeleton. It is now clear that a wide variety of cells,... (Review)
Review
Blebs are membrane structures formed by the detachment of the plasma membrane from the underlying actin cytoskeleton. It is now clear that a wide variety of cells, including cancer cells, actively form blebs for cell migration and cell survival. The expansion of blebs has been regarded as the passive ballooning of the plasma membrane by an abrupt increase in intracellular pressure. However, recent studies revealed the importance of 'cytoplasmic zoning', i.e. local changes in the hydrodynamic properties and the ionic and protein content of the cytoplasm. In this review, we summarize the current understanding of the molecular mechanisms behind cytoplasmic zoning and its role in bleb expansion.
Topics: Cytoplasm; Cytosol; Cell Membrane; Actin Cytoskeleton; Actins
PubMed: 37943501
DOI: 10.1093/jb/mvad084 -
The Journal of Cell Biology Nov 2023Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning...
Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.
Topics: Animals; Male; Mice; Actin Cytoskeleton; Actins; Cytoskeletal Proteins; Myosin Type II; Oocytes; Rotation; Female; Protein Serine-Threonine Kinases; Spindle Poles; Anaphase
PubMed: 37651121
DOI: 10.1083/jcb.202211029 -
Brain : a Journal of Neurology Oct 2023Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc....
Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology.
Topics: Humans; Filamins; Proteomics; Mutation; Muscular Diseases; Muscle Weakness; Carrier Proteins; Cytoskeletal Proteins
PubMed: 37163662
DOI: 10.1093/brain/awad152 -
Biochemical Society Transactions Dec 2023Plant organelles predominantly rely on the actin cytoskeleton and the myosin motors for long-distance trafficking, while using microtubules and the kinesin motors mostly...
Plant organelles predominantly rely on the actin cytoskeleton and the myosin motors for long-distance trafficking, while using microtubules and the kinesin motors mostly for short-range movement. The distribution and motility of organelles in the plant cell are fundamentally important to robust plant growth and defense. Chloroplasts, mitochondria, and peroxisomes are essential organelles in plants that function independently and coordinately during energy metabolism and other key metabolic processes. In response to developmental and environmental stimuli, these energy organelles modulate their metabolism, morphology, abundance, distribution and motility in the cell to meet the need of the plant. Consistent with their metabolic links in processes like photorespiration and fatty acid mobilization is the frequently observed inter-organellar physical interaction, sometimes through organelle membranous protrusions. The development of various organelle-specific fluorescent protein tags has allowed the simultaneous visualization of organelle movement in living plant cells by confocal microscopy. These energy organelles display an array of morphology and movement patterns and redistribute within the cell in response to changes such as varying light conditions, temperature fluctuations, ROS-inducible treatments, and during pollen tube development and immune response, independently or in association with one another. Although there are more reports on the mechanism of chloroplast movement than that of peroxisomes and mitochondria, our knowledge of how and why these three energy organelles move and distribute in the plant cell is still scarce at the functional and mechanistic level. It is critical to identify factors that control organelle motility coupled with plant growth, development, and stress response.
Topics: Organelles; Actin Cytoskeleton; Peroxisomes; Chloroplasts; Mitochondria; Microtubules
PubMed: 37975429
DOI: 10.1042/BST20221093 -
Cell Jun 2024Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell...
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin β-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Topics: Animals; Humans; Mice; Actin Cytoskeleton; Cell Adhesion; CHO Cells; Cricetulus; Integrins; Ligands; Protein Binding; Protein Conformation; Signal Transduction; Single Molecule Imaging; Talin
PubMed: 38772370
DOI: 10.1016/j.cell.2024.04.049 -
Developmental Cell Apr 2024The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation....
The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation. Surprisingly, we identify that mouse oocytes lacking the Arp2/3 complex undergo cortical actin remodeling upon division, followed by cortical contractions that are unprecedented in mammalian oocytes. Using genetics, imaging, and machine learning, we show that these contractions stir the cytoplasm, resulting in impaired organelle organization and activity. Oocyte capacity to avoid polyspermy is impacted, leading to a reduced female fertility. We could diminish contractions and rescue cytoplasmic anomalies. Similar contractions were observed in human oocytes collected as byproducts during IVF (in vitro fertilization) procedures. These contractions correlate with increased cytoplasmic motion, but not with defects in spindle assembly or aneuploidy in mice or humans. Our study highlights a multiscale effect connecting cortical F-actin, contractions, and cytoplasmic organization and affecting oocyte quality, with implications for female fertility.
Topics: Humans; Female; Animals; Mice; Spindle Apparatus; Oocytes; Cytoplasm; Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Actins; Meiosis; Mammals
PubMed: 38387459
DOI: 10.1016/j.devcel.2024.01.027 -
Annual Review of Physiology Feb 2024Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular... (Review)
Review
Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.
Topics: Humans; Actins; Muscle, Skeletal; Myocardium; Myosins; Actin Cytoskeleton; Calcium
PubMed: 37931167
DOI: 10.1146/annurev-physiol-042222-022728 -
The Journal of Cell Biology Mar 2024In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane...
In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.
Topics: Humans; Actin Cytoskeleton; Autophagosomes; Dynactin Complex; Dyneins; GTPase-Activating Proteins; Lysosomes; rab GTP-Binding Proteins; Sorting Nexins
PubMed: 38323995
DOI: 10.1083/jcb.202306040