Actin Self Organization
Self organization of the actin arrays and arising of cellular chirality
During cell morphogenesis, the dynamic cell form and actin cytoskeleton self-organization exist in a feedback loop. Actin cytoskeleton remodelling alters cell shape, and vice versa. For example, in cultured fibroblasts, polarization of cell shape is accompanied by self-organization of the actin cytoskeleton into an array of actomyosin stress fibers that are oriented predominantly along the long axis of the cell.
We aim to address actin cytoskeleton self-organization under a condition that constrains cell shape and ensures the cell is maintained with an isotropic morphology. This means the cytoskeletal dynamics are not influenced by changes to cell shape. To achieve this, we study the dynamics of the actin cytoskeleton in human fibroblast confined to circular fibronectin islands.
We have learnt that the actin cytoskeleton self-organizes into distinct patterns over time. This begins at the cell edge with the formation of actin fibers growing radially from peripheral focal adhesions. Subsequently, transverse fibers that are oriented perpendicularly to the radial fibers emerge, and move towards the cell center. This radial pattern is followed by a chiral pattern that is characterized by a synchronous tilting of the radial fibers, resulting in a tangential component of transverse fiber movement. The overall motion of the actin fibers that form this chiral pattern is reminiscent of a swirling fluid flow. Remarkably, in our system, the swirling process has a definite handedness.
Global organization of myosin-II in cells
Actin stress fibers are important structures in non-muscle cells. Despite being identified long ago, it remains unclear how stress fiber sarcomeres are organized in non-muscle cells. Myosin II, which is the actin motor protein, facilitates the contractility of actin stress fibers. Myosin II exhibits a periodic sarcomere-like organization in stress fibers.
The image is captured by structured illumination microscopy, 100x objective.
REF52 cells are expressed with myosin light chain (MLC) eGFP and α−actinin mCherry.
MLC is in green, and α−actinin is in hot-red. Myosin II is revealed in the organization of stack.
Myosin II stack and α−actinin are in complementary distribution along stress fibers.
The scale bar is 5 μm.
Using REF52 cells and structured illumination microscopy (SIM), we found that myosin II doublets organize into stacks that are arranged perpendicular to actin filaments. α-actinin is present between the myosin II stacks. Whereas the external spacing of myosin II stacks decreases from the cell edge to the cell center in actin transverse arcs, it is almost uniform in actin stress fibers. Myosin II doublets can assemble to stack inside the cell or near cell edge. The speed decreases when myosin filaments move from the transition zone to the lamellum in the leading edge. Investigation of the myosin stack self-organization is ongoing and will allow us to understand the sarcomeric organization of stress fibers.
The role of formins and the actin cytoskeleton in microtubule-based intracellular trafficking
Understanding the role of the mammalian formin family in the regulation of organelle structure and intracellular trafficking
Intracellular trafficking is defined as the transport of molecules from the donor compartment to the target compartment. This process requires the formation of membrane bound carriers from the donor compartment, movement of these carriers along cytoskeleton tracks and then fusion of these transport carriers at the target membrane. This process is highly dynamic and is regulated by a number of cellular machinery including microtubule and actin filaments. The role of microtubule in regulating organelle structure and intracellular trafficking has been extensively studied. In contrast, the role of actin and their regulatory proteins in regulating organelle structure and membrane transport is not well understood.
Recently, we and others have identified members of a family of proteins known as formin that are involved in the regulation of the Golgi structure [1-4]. The formin family of proteins can stimulate both nucleation and elongation of actin filaments. A common theme emerging from these recent studies is that modifications in actin polymerization may result in changes in Golgi structure and perhaps in intracellular trafficking. Alterations in intracellular trafficking induced by formins would likely impact upon physiological processes such as the secretion of cytokines, antigen presentation, and the uptake of nutrients. Here, a custom-made siRNA library targeting all mammalian formins will be used to screen for defects in Golgi structure and intracellular trafficking. A number of cargos and organelle markers will be examined by microscopy (Figure 1). We aim to understand the role of different formins in regulating the different aspects of intracellular trafficking such as fission of transport carriers, their movement along microtubules and fusion with the target membrane.
A role for the formin mDia1 in regulating microtubule based movement of organelles and vesicles in mammalian cells
We have recently identified the formin mDia1 as an important regulator of Golgi structure . A change in either the level of mDia1 or activity of mDia1 resulted in profound changes in the structure of the Golgi (Figure 2). In addition, the fission of Golgi derived Rab6 positive carriers was also affected by changes in mDia1 activity, suggesting that mDia1 functions in the regulation of both Golgi structure and fission of transport carriers. However, the role of the RhoA-mDia1 pathway in regulating the movement of organelles and carriers along the microtubule track is unclear. Recently, we have developed a system to image cells expressing low levels of a GFP-tagged cargo construct at 33 frames sec-1, which allows us to track all particles automatically.
We have also developed a set of image analysis programs that allow us to analyze the tracks and provide quantitative data on the speed, duration and direction of particle movement. Using this system, we have been able to visualize GFP-tagged cargo by fluorescence microscopy at ~33 frames sec-1. We have preliminary data indicating that changes in the level and activity of mDia1 can affect the movement of both endocytic and exocytotic carriers. Overexpression of activated mDia1 reduced the movement of both Rab5 (endocytic carriers), Rab 6 and CCL2 (exocytic carriers) in transfected cells. This process is dependent on actin polymerization as a low concentration of actin deploymerization agent can rescue movement in transfected cells, suggesting that formin derived actin polymerization is involved in the regulation of microtubule based transport. We hypothesis that actin polymerization activity of formin is required for the activity of microtubule based motors such as kinesin and dynein.
Formin-driven polymerization in vitro
Mechanical force is a key regulator of actin organization and dynamics. Many actin regulating proteins are involved in the processes of mechanotransduction. Formin, a family of barbed end tracking proteins, functions to generate long actin fibers by promoting processive elongation. It has long been proposed that formin proteins may mediate tension induced actin polymerization. However, probably due to the lack of proper techniques, this hypothesis has not been well addressed. Here, we explore the effects of tension on formin mediated actin polymerization at a single molecule level. We first developed several methods to manipulate growing actin filaments capped by formin. These methods have their unique advantages and all together cover the requirements from high efficiency to high fidelity. Now we are investigating the influence of tension on critical concentration and polymerization rates. Some preliminary results show that the applied forces may shift the equilibrium of actin dynamics towards polymerization. Although the question is still being investigated, the knowledge of mechanical response will remodel our understanding of formin's roles in mechanosensing and the regulation of actin dynamics by force.
Figure 1. Model of force in promoting formin mediated actin polymerization. a (1-3) Formin FH2 dimer processively tracks the barbed ends during polymerization. b (1-3) Stretching force applied on FH2 dimer may promote actin polymerization by facilitating translocation and lowering energy barrier.
Figure 2. Strategy of experiments. Both of the formin dimer and actin filament are anchored for the application of pulling force. Actin incorporation happens at the barbed end. Change of polymerization rates will be quantified in high resolution.
Figure 3. An actin filament anchored in between a large bead coated with mDia1 (immobilized on surface) and a small bead that binds to the side. The filament is elongating in the presence of G-actin.
Filopodia effects of mechanical forces
Filopodia are dynamic cellular protrusions composed of actin filament bundles. They are important in many cellular activities including cell migration, neuronal growth, cell-cell junction formation and others. Unconventional myosin X (MYO10) is a powerful inducer of filopodia formation and elongation once it undergoes over-expression. MYO10 is present at the filopodia tip as a bright puncta and remains there as the filopodia extend and retract. The exact mechanism by which MYO10 induces filopodia formation remains unclear. Members of the formin family have been found in filopodia, though the exact role they play in filopodia dynamics remains controversial.
Formin DIAPH3 at myosin 10 induced filopodia of HeLa cells. Snapshots of time-lapse confocal movie of HeLa cells with GFP myosin 10 and Cherry DIAPH3 overexpression. Line profiles at each time point of the myosin 10 (green) and the DIAPH3 (red) fluorescence intensities (arbitrary units) along the white dashed line shows that DIAPH3 stays close to the tip of the filopodia during time of acquisition.
We showed that MYO10 induced filopodia formation is highly dependent on formins. We found that at least a few formins, namely DIAPH1, DIAPH3, FMNL2 and DAAM1, were present in MYO10 induced filopodia of HeLa cells. The formin inhibitor SMIFH2 dramatically decreased the number and length of MYO10 induced filopodia. The phenotype observed after SMIFH2 exposure is consistent with knockdown experiments on individual formins. Low doses of SMIFH2 caused gradual changes in the dynamics of MYO10 enriched puncta at the filopodia tip. We observed numerous MYO10 positive patches being pinched out of the MYO10 puncta at filopodia tip. Applying ROCK inhibitor Y-27632 to SMIFH2 pre-treated cells blocked the fast rearward movement of MYO10 patches, suggesting possible involvement of the molecular motor myosin II pathway in MYO10 induced filopodia dynamics. We demonstrated that myosin II was physically present at the base region of MYO10 induced filopodia. Our findings show, therefore, that formins are involved in MYO10 induced filopodia dynamics and that this involvement might be orchestrated by acto-myosin II mechanosensing machinery.
We study force-induced elongation of filopodia by coupling immobilized by optical tweezers fibronectin-coated latex bead to the tip of MYO10 induced filopodia. We found that the rate and time of attachment to the tip of filopodia are dramatically different for control and formin knockdown cells. Further investigation of the system requires for establishing force-elongation relationship in filopodia dynamics. (In collaboration with Dr Artem Efremov from Yan Jie lab.)
Formin-driven assembly of perinuclear actin
The ability of cells to sense the physical microenvironment and respond to mechanical cues is critical for biological functions and survival. The actin cytoskeleton, which connects focal adhesions to the nucleus, plays a central role in cellular mechanotransduction.
Time lapse images (EGFP-Lifeact and phase contrast) showing the transient assembly of perinuclear actin upon mechanical stimulation by micropipette.
Recently we have found that an intense mechanical stimulation, such as pushing the cell at its periphery with a micropipette triggers immediate actin cytoskeleton reorganization, namely, assembly of a transient F-actin ring around the nuclear envelope (Image and Video, labelled by EGFP-Lifeact). Calcium signalling plays a key role in this process, as removing extracellular calcium by EGTA treatment completely blocks actin ring assembly. Further, we found that addition of the calcium ionophore A23187 induces the same response as mechanical stimulation. Our current work focuses on revealing the mechanism underlying the formation of the perinuclear actin ring. (In collaboration with Dr Li Qingsen from Shivashankar lab.)
Signaling to podosomes
Podosomes are integrin-mediated adhesions formed by cells of the monocytic lineage and Src-transformed fibroblasts. Recently, we showed that fibroblasts, which typically do not form podosomes develop podosome-like adhesions when grown on fluid, RGD-functionalized lipid bilayer that lacks traction force( Yu et al 2013). Podosomes are multiple micron-sized radially symmetrical structures consisting of a central actin core associated with an “adhesive ring” containing the integrins and associated plaque proteins. Actin polymerization mediated by Arp2/3 complex is critically important for podosome formation, while upstream signaling pathways involving Rho GTPases(in particular Cdc42) are known, otherwise they are poorly understood.
Actin core(red) and vinculin ring(green) of podosomes in THP1
cell treated with 1 ng/ml of TGFβ1. Scale bar, 5μm.
Our work involves the study of small GTPases(ARFs and Rabs) in regulating the assembly and size of podosomes. In addition, we are interested in understanding the structural organization of existing and newly found components in podosomes.
Signaling regulation of focal adhesion mechanosensitivity
Matrix rigidity is an important physical cue that can cause changes in cell morphology, cell migration and cell differentiation. However, the mechanism of how cells test the rigidity of the substrate is still unclear. Many different signaling pathways have been implicated in cell rigidity sensing. Using nanofabrication technology, we have developed a new tool to investigate this: a system of submicron PDMS pillars. We are using this system to quantitatively test whether particular kinases are involved in cell rigidity sensing.
Focal adhesion turnover regulation by matrix metalloproteinases
Focal adhesions, which serve as the mechanical linkage between cell and extracellular matrix, are critical structures for several biological events such as cell migration and cancer metastasis. Focal adhesions undergo dynamic turnover in response to intracellular and extracellular signals, but the mechanisms underlying this spatio-temporal control have been elusive. The microtubule cytoskeleton has been proposed to facilitate focal adhesion turnover by trafficking ‘signals’ toward focal adhesions.
Polymerization and depolymerization of microtubules induce focal adhesion turnover. HT1080 cells are treated without (left) or with (middle) a microtubule depolymerizing drug, nocodazole and then washed out (right). Cells are fixed and stained with tubulin (green) and paxillin (red) antibodies. Scale bar = 5μm.
We hypothesize that dynamic structural interactions between microtubules and focal adhesions mediates intracellular trafficking of matrix metalloproteases to focal adhesions, which in turn induces degradation of extracellular matrix, leading to modification of the physical tension applied to focal adhesions. We aim to assess the molecular and physical basis of focal adhesion turnover by optical approaches such as live imaging, super-resolution microscopy and traction force microscopy.