The Spin-Grower: A Machine for Rapid Layer-by-Layer Assembly of Nanostructured Materials

ME450 Capstone Design and Manufacturing Experience: Winter 2008Layer-by-layer (LBL) assembly is a well-established method of producing multilayered nanostructured materials. In Professor Nicholas A. Kotov’s lab at the University of Michigan, LBL assembly is often accomplished via a dip-coating process, which is time consuming and often performed on unreliable equipment. Spin-assisted LBL assembly has the potential to reduce the fabrication time of nanostructured materials by an order of magnitude and increase the quality of the films. The purpose of this project is to design and produce a spin-assisted LBL assembly prototype using a spin-coater and an automated fluid delivery system for the production of a variety of different nanocomposites.Prof. Nicholas A. Kotov, Prof. John Hart, and Paul Podsiadlohttp://deepblue.lib.umich.edu/bitstream/2027.42/58685/1/me450w08project17_report.pd

Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of Src

It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET) method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor) and YPet (a variant yellow fluorescent protein as an acceptor) of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms) of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF) induced Rac activation at a much later time (>30 sec). There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17) was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces via integrins might be fundamentally different from those of growth factors

Soft Substrates Promote Homogeneous Self-Renewal of Embryonic Stem Cells via Downregulating Cell-Matrix Tractions

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions

Rapid Rac GTP-ASE activation in live cells by mechanical stress is independent of Src

It is well-known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET) method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor) and YPet (a variant yellow fluorescent protein as an acceptor) of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms) of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF) induced Rac activation at a much later time (>30 sec). It is known that PDGF-induced Rac activation depends on Src activity. There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17) was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces might be fundamentally different from those of growth factors

Biophysical and gene expression change in living cells by force-induced mechanotransduction

Within the past decade, there has been abounding scientific evidences supporting the notion that mechanical forces are crucial in regulating the physiologic functions of cells and tissues. The importance of engineering principles in studying the biological behavior of cells is no longer in question. Instead, much research is now focused on how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level - a process known as mechanotransduction. This work uses both engineering and biological principles to investigate the different biophysical and gene expression changes of individual cells in response to exogenous forces. We attempt to unravel the mechanism at which forces are transmitted from the apical surface of the cell in to the nucleus. The work presented here provides the first unequivocal evidence that a local surface force can directly alter nuclear functions without intermediate biochemical cascades. We show that a local dynamic force via integrins results in direct displacements of coilin and SMN proteins in Cajal bodies and direct dissociation of coilin-SMN associated complexes. Fluorescence resonance energy transfer changes of coilin-SMN depend on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in Cajal bodies exhibit different magnitudes of fluorescence resonance energy transfer. Dynamic cyclic force induces tiny phase lags between various protein pairs in Cajal bodies, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression. We further extend our study to mouse embryonic stem cells (ESCs). Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. We demonstrate that forces transmitted through different natural extracellular matrix proteins or cell-cell adhesion molecules such as fibronectin, laminin or E-cadherin, have different effects on cell spreading, cell stiffness, Oct3/4 gene expression, and cell proliferation rate. Surprisingly, it was also observed that mouse ESCs do not stiffen when substrate stiffness increases. These cells do not increase spreading on more-rigid substrates either. However, ESCs do increase their basal tractions as substrate stiffness increases. ESCs therefore exhibit mechanical behaviors distinct from those of mesenchymal stem cells and of terminally differentiated cells, and decouple its apical cell stiffness from its basal tractional stresses during the substrate rigidity response. We further elucidate how mechanical forces influence the differentiation of ESCs into spatially organized endoderm, mesoderm, and ectoderm germ layers. ESCs cultured within 3D soft fibrin gels in the absence of Leukemia Inhibitory Factor (LIF) promotes in vivo tissue morphogenesis during vertebrate gastrulation. The results presented demonstrate that mechanical forces play different roles in different force transduction pathways to shape early embryogenesis

Rapid Rac activation despite extraction of cholesterol from the plasma membrane.

<p>(A) Representative time-lapse emission ratio images of a SYF−/− MEF cell treated with 10 mM of methyl-β-cyclodextrin (MβCD) for 15 min to selectively extract cholesterol from the plasma membrane before the application of stress. Inset shows the enlarged area of the cell periphery where rapid activation of Rac is observed. The left inset panel shows the brightfield image of the cell with a magnetic bead (black dot) bound to the apical surface. (B) Normalized emission ratio time course of SYF −/− MEFs in response to 17.5 Pa stress pretreated with methyl-β-cyclodextrin (MβCD). This shows that the activation of Rac occurs independent of the integrity of lipid rafts at the plasma membrane. (n = 4 cells, mean +/− s.e.).</p

Rapid Rac activation in response to a local mechanical stress.

<p>(A) A 4.5-µm RGD-coated ferromagnetic bead was attached to the apical surface of the cell (black dot is the bead) for 15 min to allow integrin clustering and formation of focal adhesions around the bead. The bead was magnetized horizontally and subjected to a vertical magnetic field (step function) which applies a mechanical rotational stress (apparent average stress = 17.5 Pa) to the cell. A genetically encoded, CFP-YPet cytosolic Rac reporter was transfected into the smooth muscle cells following published procedures. The cytosolic Rac reporter was uniformly distributed in the cytoplasm (YPet fluorescence; white arrow indicates bead movement direction). The stress application induced rapid changes (<0.3 s) in FRET of the Rac reporter at discrete, distant sites at the cell periphery (the focal plane is ∼1 µm above cell base), indicating rapid Rac activation (see Insets). Images are scaled and regions of large FRET changes (strong Rac activity) are shown in red/yellow. Scale bar  = 10 µm. (B) Time-lapse images of Rac activation at the cell periphery after addition of PDGF (10 ng/ml) shows that activation of Rac in a representative cell by soluble factor PDGF is slow. Note that significant Rac activation occurred only at 0.5–1 min after PDGF treatment. Insets are enlarged areas showing Rac activation at the cell periphery. Scale bar  = 10 µm. (C) Normalized emission ratio of FRET as a function of stress application duration for the Rac biosensor (control) and its mutant form(RacN17) (Control, n = 6 cells; RacN17, n = 3 cells; mean +/− s.e.). A representative cell was shown in A with the Rac biosensor. (D) Normalized YPet/CFP emission ratio time courses of the Rac biosensor (Control) and the mutant Rac biosensor (RacN17) in response to PDGF treatment. (Control, n = 4 cells; RacN17, n = 3 cells; mean +/− s.e.).</p

Cytosketal integrity is necessary for Rac activation by stress.

<p>(A) Inhibition cytoskeletal tension with blebbistatin (Blebb, 50 µM for 30 min, n = 7 cells), disrupting F-actin with cytochalasin D (CytoD, 1 µg/ml for 15 min, n = 6 cells) or microtubules with colchicine (Colch, 10 µM for 15 min, n = 9 cells), blocks stress-induced Rac activation in SYF−/− MEFs. Mean+/−s.e. (B) A working model for rapid Rac activation by stress. A local load (magnetic bead) applied to focal adhesions leads to stress propagation along the actin bundles (red lines) without decay in magnitudes at remote sites. Rac GTPase bound to the plasma membrane at the other end of the cell are activated rapidly when stress waves reach the plasma membrane via the cytoskeleton to directly deform Rac, causing a conformational change in the enzyme. MF =  actin microfilament; MT = microtubule; IF = intermediate filament; SF = stress fiber; N = nucleus (not drawn to scale). Black dot = the magnetic bead. White arrow = magnetic moment direction of the magnetized bead. Curved black arrow = the rotational shear stress.</p