Ziya Kalay

Dynamic Organizing Principles of the Plasma Membrane that Regulate Signal Transduction: Commemorating the Fortieth Anniversary of Singer and Nicolson's Fluid-Mosaic Model

The recent rapid accumulation of knowledge on the dynamics and structure of the plasma membrane has prompted major modifications of the textbook fluid-mosaic model. However, because the new data have been obtained in a variety of research contexts using various biological paradigms, the impact of the critical conceptual modifications on biomedical research and development has been limited. In this review, we try to synthesize our current biological, chemical, and physical knowledge about the plasma membrane to provide new fundamental organizing principles of this structure that underlie every molecular mechanism that realizes its functions. Special attention is paid to signal transduction function and the dynamic aspect of the organizing principles. We propose that the cooperative action of the hierarchical three-tiered mesoscale (2-300 nm) domains--actin-membrane-skeleton induced compartments (40-300 nm), raft domains (2-20 nm), and dynamic protein complex domains (3-10 nm)--is critical for membrane function and distinguishes the plasma membrane from a classical Singer-Nicolson-type model.

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

Ultraspeed single-molecule tracking with <25-μs resolution and electron tomography show that transmembrane proteins and phospholipids in the plasma membrane hop among submicrometer compartments of the same size, probably delimited by the anchored-transmembrane-protein pickets lining the actin-based membrane-skeleton fence, once every 1–58 ms.

Confining Domains Lead to Reaction Bursts: Reaction Kinetics in the Plasma Membrane

Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity.

Archipelago architecture of the focal adhesion: Membrane molecules freely enter and exit from the focal adhesion zone

The focal adhesion (FA) is an integrin‐based structure built in/on the plasma membrane, mechanically linking the extracellular matrix with the termini of actin stress fibers, providing key scaffolds for the cells to migrate in tissues. The FA was considered as a micron‐scale, massive assembly of various proteins, although its formation and decomposition occur quickly in several to several 10 s of minutes. The mechanism of rapid FA regulation has been a major mystery in cell biology. Here, using fast single fluorescent‐molecule imaging, we found that transferrin receptor and Thy1, non‐FA membrane proteins, readily enter the FA zone, diffuse rapidly there, and exit into the bulk plasma membrane. Integrin β3 also readily enters the FA zone, and repeatedly undergoes temporary immobilization and diffusion in the FA zone, whereas approximately one‐third of integrin β3 is immobilized there. These results are consistent with the archipelago architecture of the FA, which consists of many integrin islands: the membrane molecules enter the inter‐island channels rather freely, and the integrins in the integrin islands can be rapidly exchanged with those in the bulk membrane. Such an archipelago architecture would allow rapid FA formation and disintegration, and might be applicable to other large protein domains in the plasma membrane.

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non‐raft phospholipid molecules (0.33 µm2/second), despite Bdp‐Chols probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.

Reaction kinetics in the plasma membrane

A great puzzle in science is establishing a bottom up understanding of life by revealing how a collection of molecules gives rise to a living cell that can survive, communicate, and reproduce. In the confines of physics, chemistry, or material science laboratories where it possible to study complex interactions between molecules in a well‐defined environment, our understanding of collective behavior is substantially developed. However, the environment in which molecules of a biological cell perform their functions is far from ideal or controllable. The environment inside cellular regions such as the plasma membrane is heterogeneous and dynamic, and functional molecules such as proteins are both dynamic and promiscuous, as they interact with countless other molecules. This makes it extremely challenging to grasp the inner mechanism of the cells, both experimentally and theoretically. On the bright side, this presents scientists with a colorful playground that waits to be explored: the mesoscopic world inside the cell. This review covers some of the recent experimental and theoretical developments in the study of molecular interactions in the plasma membrane, viewed as a heterogeneous medium where the number of reactants can be small, sometimes countable, and its implications for biological function.

Fundamental and functional aspects of mesoscopic architectures with examples in physics, cell biology, and chemistry

How small can a macroscopic object be made without losing its intended function? Obviously, the smallest possible size is determined by the size of an atom, but it is not so obvious how many atoms are required to assemble an object so small, and yet that performs the same function as its macroscopic counterpart. In this review, we are concerned with objects of intermediate nature, lying between the microscopic and the macroscopic world. In physics and chemistry literature, this regime in-between is often called mesoscopic, and is known to bear interesting and counterintuitive features. After a brief introduction to the concept of mesoscopic systems from the perspective of physics, we discuss the functional aspects of mesoscopic architectures in cell biology, and supramolecular chemistry through many examples from the literature. We argue that the biochemistry of the cell is largely regulated by mesoscopic functional architectures; however, the significance of mesoscopic phenomena seems to be quite underappreciated in biological sciences. With this motivation, one of our main purposes here is to emphasize the critical role that mesoscopic structures play in cell biology and biochemistry.

Fragmentation of random trees

We study fragmentation of a random recursive tree into a forest by repeated removal of nodes. The initial tree consists of N nodes and it is generated by sequential addition of nodes with each new node attaching to a randomly-selected existing node. As nodes are removed from the tree, one at a time, the tree dissolves into an ensemble of separate trees, namely, a forest. We study statistical properties of trees and nodes in this heterogeneous forest, and find that the fraction of remaining nodes m characterizes the system in the limit N --> infty. We obtain analytically the size density phi_s of trees of size s. The size density has power-law tail phi_s ~ s^(-alpha) with exponent alpha=1+1/m. Therefore, the tail becomes steeper as further nodes are removed, and the fragmentation process is unusual in that exponent alpha increases continuously with time. We also extend our analysis to the case where nodes are added as well as removed, and obtain the asymptotic size density for growing trees.

Effects of confinement on the statistics of encounter times: exact analytical results for random walks in a partitioned lattice

We study the effects of temporarily and permanently confining domains on the statistics of first-passage times in finite lattices in one and two dimensions. We present exact results for the mean and variance of the first-passage time between arbitrary sites in the following: (1) a finite one-dimensional lattice partitioned into temporarily confining domains and (2) a finite two-dimensional lattice with reflecting boundaries for a single random walker and an immobile target. In the one-dimensional case, we also present the full first-passage time distribution via numerical inversion of Laplace transforms.

Exact Green's functions for a Brownian particle reversibly binding to a fixed target in a finite, two-dimensional, circular domain

Despite the apparent need to study reversible reactions between molecules confined to a two-dimensional space such as the cell membrane, exact Green?s functions for this case have not been reported. Here we present exact analytical Green?s functions for a Brownian particle reversibly reacting with a fixed reaction center in a finite two-dimensional circular region with reflecting or absorbing boundaries, considering either a spherically symmetric initial distribution or a particle that is initially bound. We show that Green?s function can be used to predict the effect of measurement uncertainties on the outcome of single-particle/molecule-tracking experiments in which molecular interactions are investigated. Hence, we bridge the gap between previously known solutions in one dimension (Agmon 1984 J. Chem. Phys. 81 2811) and three dimensions (Kim and Shin 1999 Phys. Rev. Lett. 82 1578), and provide an example of how the knowledge of Green?s function can be used to predict experimentally accessible quantities.