Manorama Tewari

Shape effects of filaments versus spherical particles in flow and drug delivery

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.

Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

Introduction Tissues can be soft like brain, bone marrow, and fat, which bear little mechanical stress, or stiff like muscle, cartilage, and bone, which sustain high levels of stress. Systematic relationships between tissue stiffness, protein abundance, and differential gene expression are unclear. Recent studies of stem cells cultured on matrices of different elasticity, E, have suggested that differentiation is mechanosensitive, but the molecular mechanisms involved in particular tissues remain elusive. Tissue micromechanics correlate with abundance of collagens and nuclear lamins, which influence cell differentiation. (Left) Collagen and lamin-A levels scale with E, consistent with matching tissue stress to nuclear mechanics. (Right) Matrix stiffness in tissue culture increases cell tension and stabilizes lamin-A, regulating its own transcription and that of stress fiber genes, enhancing differentiation. RA, retinoic acid, i.e., vitamin A; RARG, YAP1, and SRF, transcription factors. Methods We developed quantitative mass spectrometry algorithms to measure protein abundance, stoichiometry, conformation, and interactions within tissues and cells in relation to stiffness of tissues and extracellular matrix. Manipulations of lamin-A levels with small interfering RNA, overexpression, and retinoic acid or antagonist were applied to stem cells cultured on different matrices to assess lamin-A’s role in mechanosensitive differentiation. To characterize molecular mechanisms, promoter analyses, transcriptional profiling, and localization of transcription factors were complemented by measurements of nuclear mechanics and by modeling of the core gene circuit. Results Proteomic profiling of multiple adult solid tissues showed that widely varied levels of collagens in extracellular matrix and of lamin-A in nuclei followed power-law scaling versus E. Scaling for mechanoresponsive lamin-A conformed to predictions from polymer physics, whereas lamin-B’s varied weakly. Tumor xenograft studies further demonstrated that matrix determined tissue E, whereas lamin-A levels responded to changes in E. In tissue culture cells, both lamin-A conformation and expression were mechanosensitive, with phosphorylation and turnover of lamin-A correlating inversely with matrix E. Lamin-A knockdown enhanced mesenchymal stem cell differentiation on soft matrix that favored a low-stress, fat phenotype. Lamin-A overexpression or transcriptional induction with a retinoic acid (RA) antagonist enhanced differentiation on stiff matrix toward a high-stress, bone phenotype. Downstream of matrix stiffness, the RA pathway regulated lamin-A transcription, but feedback by lamin-A regulated RA receptor (RARG) translocation into nuclei. High lamin-A levels physically impeded nuclear remodeling under stress but also coregulated other key factors. These factors included both serum response factor (SRF), which promoted expression of stress fiber–associated proteins involved in differentiation, and a Hippo pathway factor (YAP1) involved in growth. Discussion The characteristic stress in normal tissue favors collagen accumulation and a characteristic stiffness that cells transduce through nuclear lamin-A to enhance tissue-specific differentiation. Tension-inhibited turnover of rope-like filaments of lamin-A provides sufficient mechanochemical control of a core gene circuit to explain the steady-state scaling of lamin-A with E. High lamin-A physically stabilizes the nucleus against stress and thereby stabilizes the nuclear lamina and chromatin, with implications for epigenetic stabilization and limiting of DNA breaks. Moreover, lamin-A levels directly or indirectly regulate many proteins involved in tissue-specific gene expression, and, because lamin-A levels can vary by a factor of 10 or more downstream of tissue mechanics, an important fraction of tissue-specific gene expression depends on tissue mechanics, which changes in development, injury, and many diseases. Lamins and Tissue Stiffness Microenvironment can influence cell fate and behavior; for example, extracellular matrix (ECM) stiffness increases cell proliferation, and ECM rigidity induces disorders in tissue morphogenesis by increasing cell tension. Swift et al. (1240104; see the Perspective by Bainer and Weaver) used proteomics to identify molecules that are mechanical sensors for tissue elasticity in the nucleus and discovered that expression of lamin-A levels apparently functions as a “mechanostat.” Tissues that need to remain stiff under stress rely on lamin-A to keep the cell nucleus whole. [Also see Perspective by Bainer and Weaver] Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

γ-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion

The functions of γ-sarcoglycan (γSG) in normal myotubes are largely unknown, however γSG is known to assemble into a key membrane complex with dystroglycan and its deficiency is one known cause of limb-girdle muscular dystrophy. Previous findings of apoptosis from γSG-deficient mice are extended here to cell culture where apoptosis is seen to increase more than tenfold in γSG-deficient myotubes compared with normal cells. The deficient myotubes also exhibit an increased contractile prestress that results in greater shortening and widening when the cells are either lightly detached or self-detached. However, micropipette-forced peeling of single myotubes revealed no significant difference in cell adhesion. Consistent with a more contractile phenotype, acto-myosin striations were more prominent in γSG-deficient myotubes than in normal cells. An initial phosphoscreen of more than 12 signaling proteins revealed a number of differences between normal and γSG–/– muscle, both before and after stretching. MAPK-pathway proteins displayed the largest changes in activation, although significant phosphorylation also appeared for other proteins linked to hypertension. We conclude that γSG normally moderates contractile prestress in skeletal muscle, and we propose a role for γSG in membrane-based signaling of the effects of prestress and sarcomerogenesis.

Upregulation of paxillin and focal adhesion signaling follows Dystroglycan Complex deletions and promotes a hypertensive state of differentiation.

Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG(-/-) mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG(-/-) muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.

Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics.

Force‐bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability—as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense‐mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin “nano”‐constructs based on multiexon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with liquid chromatography tandem mass spectrometry (LC‐MS/MS) which can detect tryptic peptides from 53 of dystrophins 79 exons; equivalent information from immunodetection would require 53 different high‐specificity antibodies. Folding predictions for the nano‐constructs reveal novel helical bundle domains that arise out of exon‐deleted “linkers,” while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an atomic force microscope nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multiexon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon‐skipped designs.

Mechanical signaling coordinates the embryonic heartbeat

Significance There is a mounting body of evidence that physical forces induce biochemical changes. Here, we suggest that the early embryonic heart provides a striking illustration of the importance of mechanics in living matter. Whereas adult hearts use electrical signaling to coordinate the heartbeat, we propose that embryonic hearts use mechanical signaling. We model the embryonic heart as mechanically excitable tissue, with cardiac myocytes that are triggered to contract under strain. Such contractions exert strains on nearby cells and induce further contraction, thus propagating the signal through the heart. This simple model captures key features observed in the heartbeat of stiffness-modified embryonic hearts that cannot be explained by standard electrochemical signaling and yields predictions that we confirm with experiments. In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats.