Milan Mrksich

Geometric cues for directing the differentiation of mesenchymal stem cells.

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

Micropatterned Surfaces for Control of Cell Shape, Position, and Function

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self‐assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer‐scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape. Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth. Data are presented which show how micropatterned substrates were used to definitively test this hypothesis. Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function. The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell−ECM interactions.

Peptide chips for the quantitative evaluation of protein kinase activity.

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels–Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH2 on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.

GEOMETRIC CONTROL OF SWITCHING BETWEEN GROWTH, APOPTOSIS, AND DIFFERENTIATION DURING ANGIOGENESIS USING MICROPATTERNED SUBSTRATES

SummaryPast studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 µm2 spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 µm2 failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-µm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 µm2. Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-µm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 µm2), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis—growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.

Carbohydrate Arrays for the Evaluation of Protein Binding and Enzymatic Modification

This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of beta-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures.

A surface chemistry approach to studying cell adhesion

Model substrates that present peptide and carbohydrate ligands are becoming important mechanistic tools in cell biology. This review surveys the development of self-assembled monolayers of alkanethiolates on gold as a model substrate for studies of cell adhesion. The review begins with a background that illustrates the opportunity for using tailored substrates in biology and then addresses the characteristics that make monolayers well-suited for these studies. The review concludes with a discussion of recent work that is developing dynamic substrates wherein the activity of immobilized ligands can be modulated in real time.

Patterning self-assembled monolayers using microcontact printing : a new technology for biosensors ?

Self-assembled monolayers (SAMs), formed upon the adsorption of ω-substituted alkanethiols on the surface of gold, allow control of the properties of a surface on the molecular scale. A new technique — microcontact printing (μCP) — can pattern the formation of SAMs, with dimensions on the micron scale. The convenience, low cost, and widespread application offered by SAMs and μCP make this combination of techniques especially suitable for producing and patterning surfaces relevant to biosensors.

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

Circadian Clock NAD+ Cycle Drives Mitochondrial Oxidative Metabolism in Mice

Introduction The circadian clock is a transcriptional oscillator that is thought to couple internal energetic processes with the solar cycle. Circadian oscillation in activity of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD+) biosynthesis, feeds back to regulate activity of the deacetylase SIRT1 and transcription of genes encoding core clock components. Despite evidence that NAD+-dependent enzymes are important in fasting and oxidative metabolism, it is not known how the circadian cycle might affect this process. We investigated the role of clock control of NAD+ in mitochondrial dynamics and energy production. Circadian regulation of NAD+ biosynthesis synchronizes mitochondrial bioenergetics with the light-dark cycle. The core molecular clock is a transcription-translation oscillator composed of activators (CLOCK/BMAL1) that induce transcription of their own repressors (PER/CRY). Clock control of expression of the NAD+ biosynthetic enzyme NAMPT generates 24-hour variation of activity of the mitochondrial deacetylase SIRT3 and oxygen consumption. Rhythmic NAD+ oscillation couples mitochondrial bioenergetics with the light-dark cycle. Methods We determined the circadian variation in mitochondrial function by examining the adaptive response to fasting in liver of wild-type and circadian mutant mice. Quantitative analyses of NAD+ biosynthesis, lipid and glucose oxidation, and acetylation of mitochondrial proteins were performed across the circadian cycle in circadian mutant mice and in cell-based systems. Proteins displaying increased acetylation in Bmal1 mutant liver were identified by mass spectrometry, and SIRT3 activity was evaluated using label-free self-assembled monolayer and matrix desorption ionization (SAMDI) mass spectrometry in liver lysate from Bmal1 and Sirt3 knockout mice. The role of NAD+ deficiency in SIRT3 activity, mitochondrial protein acetylation, lipid oxidation, and oxygen consumption was evaluated after intraperitoneal administration of the NAD+ precursor NMN to raise NAD+ levels in Bmal1 mutant and wild-type mice. Results Lipid oxidation and mitochondrial protein acetylation exhibited circadian oscillations that corresponded with the clock-driven NAD+ cycle in mouse liver. Rhythmic NAD+ and oxidative cycles were self-sustained in fasted mice and in C2C12 myotubes, demonstrating clock control of mitochondrial function even when nutrient state remained constant. Transcription of glycolytic genes was antiphasic to lipid oxidation rhythms, and glycolytic gene expression and lactate production were increased in Bmal1–/– fibroblasts, whereas the converse occurred in Cry1–/–;Cry2–/– mutants. Lack of Bmal1 in liver led to decreased SIRT3 activity and increased mitochondrial protein acetylation, resulting in reduced function of oxidative enzymes. Finally, NAD+ supplementation with NMN restored protein deacetylation of SIRT3 targets and enhanced mitochondrial function in circadian mutant mice. Discussion Mitochondria are central to energy homeostasis in eukaryotes, and our results show that the circadian clock generates oscillations in mitochondrial oxidative capacity through rhythmic regulation of NAD+ biosynthesis. The clock thereby facilitates oxidative rhythms that correspond with the fasting-feeding cycle to maximize energy production during rest. Use of NAD+ as a central node in coupling circadian and metabolic cycles provides a rapid and reversible mechanism to augment mitochondrial oxidative function at the appropriate time in the light-dark cycle. Dinner Time! Biological clocks allow organisms to anticipate cycles of feeding, activity, and rest so that metabolic enzymes in mitochondria are ready when needed. Peek et al. (10.1126/science.1243417, published online 19 September; see the Perspective by Rey and Reddy) describe a mechanism by which the biochemical elements of the circadian clock are linked to such control of mitochondrial metabolism. The clock controls rhythmic transcription of the gene encoding the rate-limiting enzyme required for synthesis of nicotinamide adenine dinucleotide (NAD+). The concentration of NAD+ in mitochondria determines the activity of the deacetylase SIRT3, which then controls acetylation and activity of key metabolic enzymes. NAD+ also influences clock function, and thus appears to be a versatile point at which regulation of oxidative metabolism is coordinated with the daily cycles of energy consumption. The coenzyme nicotinamide adenine dinucleotide mechanistically links the circadian clock to control of energy production by mitochondria. [Also see Perspective by Rey and Reddy] Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD+) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD+ supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD+ bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.

Using electroactive substrates to pattern the attachment of two different cell populations

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.