Forces applied at the periphery of a cell causes actin remodeling around the nucleus, leading to the formation of a transient perinuclear actin rim. The formation of this actin structure is dependent on the force-induced release of Ca2+ ions in the cytoplasm, and is mediated by the formin family member, INF-2.
These findings were published in PNAS in 2015.
Shao X et al. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim. 2015. PNAS. 112(20). E2592-2601.
More information on the Shivashankar Lab.
Figure: The schematic represents the long-acting effects of locally applied forces. a) local forces (100-200 nN) are applied at the cell periphery using an AFM tip. b) application of forces triggers the release of intracellular Ca2+ ions. c) Ca2+ signaling induces actin remodeling around the nucleus via inverted formin-2 (INF-2), leading to the assembly of a transient perinuclear actin rim.
Cells respond to the physical properties of their surroundings by remodeling their cytoskeleton, mainly actin filaments, into higher order structures that can propagate forces across the cell. Numerous studies have shown how forces affect actin remodeling directly at sites of force application. However, their effects on actin filaments at distant locations remained uncharacterized until recently.
This study reveals a previously unidentified, far-acting effect of forces on actin remodeling. By using a specially designed atomic force microscopy tip to apply forces of a magnitude between 100-200 nN at the cell periphery, the researchers noted the assembly of actin filaments around the nucleus into a transient high-order structure, which they called the perinuclear actin rim. The assembly of the perinuclear actin rim was dependent on force-induced release of Ca2+ in the cytoplasm, and was found to be mediated by the actin filament nucleation and elongation factor, inverted formin-2 (INF-2).
The perinuclear actin rim is associated with two main functions: relaying mechanical signals from the cytoplasm to the nucleus, and shielding the genome from any mechanical perturbations that could reach the nucleus.
Understanding the basics
The actin cytoskeleton is physically connected to the extracellular matrix (ECM) through multiprotein complexes known as focal adhesions. Interactions between cell-surface molecules such as integrins and the actin cytoskeleton are bidirectional, with focal adhesions forming links between them. The actin cytoskeleton and the focal adhesions function as mechanosensors: they convert the strength of the adhesions and the tensile forces along the actin cytoskeleton into biochemical signals that control cell behavior. The physico-chemical properties of the ECM, such as its rigidity, topography, and chemical composition, as well as stresses propagated via focal adhesions will influence the assembly and organization of the actin cytoskeleton.
Formins promote the elongation of pre-existing actin filaments by removing barbed end capping proteins and forming a sleeve around actin subunits. Formins are also capable of actin nucleation, which is the de novo assembly of actin filaments from actin monomers. Activated formins exist as dimers and form a donut-shaped complex around terminal actin subunits, orienting themselves toward the barbed (+) end of actin filaments. The FH2 domain facilitates binding to the growing filament; this requires removing capping proteins from the barbed ends and preventing re-capping in order to allow continued growth of actin filaments. Ena/VASP proteins support formin-mediated filament elongation by tethering the filaments near sites of active actin assembly. Each formin monomer then binds and captures profilin proteins through its FH1 domain, which are already bound to G-actin monomers. The monomers are then added to the growing actin filament.
Cytoskeletal filaments- actin filaments, intermediate filaments, and microtubules, bridge the nucleus to the plasma membrane, which in turn is anchored at sub-cellular sites to extracellular substrates via a plethora of proteins that form focal adhesions. The cytoskeletal filaments converge on the nucleus, where they bind directly to KASH domain proteins such as the nesprin proteins. These proteins localize on the outer nuclear membrane and are part of a larger complex known as the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex: a physical link that enables force transmission across the nuclear envelope to impact processes within the nucleus. Such transduction is possible as nesprin proteins are connected to the inner nuclear membrane through dimers of the SUN-domain adaptor proteins (Sad1p, UNC-84). These SUN proteins are in turn anchored to nuclear lamins, and chromatin, inside the nucleus. These links are emerging to be pivotal in various physiological processes including cell migration, and in the ability of cells to cope with mechanical stress.
The Study in Detail
- The effects of external forces on actin remodeling at distant locations inside the cell was assessed by applying forces (100-200 nN) at the periphery of NIH3T3 cells using a specially designed atomic force microscopy (AFM) tip. A transient actin structure assembled around the nucleus in response to forces, which was referred to as the perinuclear actin rim.
- Filamentous actin (F-actin) levels around the nucleus reached a maximum at 30 seconds following the application of forces and later returned to original levels within 2 minutes. F-actin levels nearer to the cell membrane showed a corresponding decrease with the increasing levels at the perinuclear region.
- There was a sudden release of Ca2+ ions from intracellular calcium stores in response to forces and this intracellular Ca2+ burst preceded perinuclear actin rim formation. Once the Ca2+ in the cytoplasm returned to basal levels, the actin rim around the nucleus also disappeared.
- When intracellular Ca2+ was depleted by EGTA treatment, the perinuclear actin rim failed to form; alternately treatment of NIH3T3 cells with a calcium ionophore (induces intracellular ca2+ release) was sufficient to trigger the formation of the perinuclear actin rim, even in the absence of mechanical forces. These findings suggested an indispensable role of Ca2+ signaling in the actin remodeling process.
- The formation of the perinuclear actin rim was independent of integrin signaling, which was confirmed by observations of actin rim formation in cells grown on poly-L-lysine substrates (i.e. without integrin ligands) or in cells treated with an FAK (integrin signaling component) inhibitor.
- Perinuclear actin rim formation in response to force/ionophore induced-Ca2+ stimulation was not prevented when cells were treated with inhibitors for actin regulators such as Arp2/3, Myosin-II, Cofilin, and Rho GTPase. This suggested that a different molecular pathway was responsible for the actin remodeling event.
- However, when inverted formin-2 (INF-2) was knocked out, the actin rim failed to form. INF-2 is an actin filament nucleation and elongation factor belonging to the formin family. This suggests crucial role for this protein in perinuclear actin polymerization. Moreover, INF-2 mediated actin polymerization was dependent on Ca2+ stimulation, since overexpression of INF-2 in the absence of Ca2+ release did not induce actin rim formation.
Methods and Controls used in the study
- An atomic force microscopy (AFM) tip attached to a 4.5 micrometer polystyrene bead that was controlled by a micromanipulator was used to apply forces at the periphery of NIH3T3 cells.
- Confocal microscopy was used for live imaging of cells in order to quantify the response of cells to application of local forces.
Applications and Future Directions
- The study provides insights into the physiological relevance of force/Ca2+-induced perinuclear actin remodeling: on one hand, it aids in the relay of mechanical signals from the cytoplasm to the nucleus by facilitating the nuclear transport of transcription factors; on the other hand, it functions as a physical barrier or a shield that protects the integrity of the genome during mechano-chemical instabilities within the cell.
- By linking INF-2 mediated perinuclear actin polymerization with genome function, the study provides clues regarding defects in INF-2 and the occurrence of diseases such as focal and segmental glomerulosclerosis and Charcot-Marie Tooth disease.
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