Mechanics of Motor Proteins and the Cytoskeleton The Howard lab is fascinated by the question of how small molecules like proteins, lipids and nucleotides self-assemble into cells and tissues that are thousands to millions of times larger than molecular dimensions. How do the molecules know where they should be, and whether the structures that they have made are the right size, shape and location? By combining highly sensitive techniques to visualize and manipulate individual biological molecules with theory and modeling, the Howard lab is trying to understand the interaction rules that allow molecules to work together to form highly organized yet dynamic cellular structures.
The Howard lab is approaching these questions in the context of the microtubule cytoskeleton. They are interested in the molecular properties of motor proteins and microtubule assembly regulators, especially how they operate as molecular machines to drive motion and regulate the growth and shrinkage of microtubules. In addition to biochemical and single-molecule approaches, they also study a number of cellular systems including the morphogenesis of neuronal dendrites and the motility of cilia & flagella. Specialized Terms: Microtubules, Motor proteins, MAPs, Cytoskeleton, Microtubule dynamics, Cell motility, Mitosis, the Axoneme, Optical tweezers, Single-molecule biophysics
Motor and Cytoskeletal Systems: From molecules to cells
The Howard lab combines experiments and theory to understand how the cytoskeleton drives cells shape and motion. Our research spans from single molecules, using state-of-the-art optical techniques, through to complex organismal systems that include dendrite development in Drosophila, cilia and flagella in Chlamydomonas, and neuronal polarity in Hydra.
How do cells measure length and time? How do they count? How do they sense their shape? These are some of the most important outstanding questions in cell and developmental biology. For example, how do proliferating cells know how big they are and when to stop growing and to start dividing? How do organs, such as brain and liver, ensure that they manufacture enough cells, but not too many? How do cells control the dimensions of their organelles, such as endosomes, cilia and mitotic spindles? How do cells make certain that they have exactly two copies of each chromosome, that they have just one cilium, but that dividing cells have exactly two mitotic-spindle poles? And how much energy is required to implement and control these processes?
These are difficult questions because they address “systems-level” processes whose length-scales and time-scales differ strikingly from those of the constituent molecules—the proteins, lipids and nucleic acids—that form the molecular building blocks of cells. The goal of our research is to find molecule-level answers to these systems-level questions. Just as a radio or a computer must be understood in terms of its constituent analog or digital circuits, cell and tissue morphology must be understood in terms of underlying molecular circuits. These circuits comprise networks of interacting molecules. What kinds of computations can they perform? How do they assemble? What feedback-control mechanisms operate to control the size, shape and localization of molecular assemblies? Reaction-diffusion equations have dominated our thinking about biological patterning since the seminar work of Turing in the 1950s; however, we argue that active, mechanical processes mediated by motor proteins and the cytoskeleton, may play an even more important role in morphogenesis than purely chemical mechanisms (Howard et al. 2011).
Research Approach: Quantitative experiments to test theoretical predictions
Our research focuses on the biochemistry and biophysics of the cytoskeleton, with particular emphasis on the mechanics of microtubules and microtubule-based motor proteins. On the one hand, the lab is interested in how these proteins work: i.e. how do kinesins, dyneins and microtubules act as molecular machines to convert chemical energy derived from the hydrolysis of ATP or GTP into mechanical work used to power cell motility? And, on the other hand, we are interested in the roles that microtubules and their motors play in shaping and moving cells and tissues. For example, how do the dynamic properties of microtubules shape neurons in the brain, how do motor proteins and microtubule regulators assemble the mitotic spindle and segregate the chromosomes, and how does dynein power the motility of cilia and flagella?
Our approach is to combine measurement and theory (Howard 2014). The challenge is that the complexity of biological systems makes quantitative measurements and their interpretation exceedingly difficult. The key to circumventing these difficulties is the use of single-molecule techniques, in whose development our lab played an important role (e.g. Howard et al. 1989). Using single-molecule optical techniques, the interactions between the individual motor and cytoskeletal molecules can be characterized in vitro and in vivo. These interactions constitute a form of mechanical signaling (Howard 2009). We then use theory, primarily from statistical physics and non-linear dynamics, to predict how the individual interactions lead to the collective behavior of ensembles of molecules (see e.g. Sartori et al. 2016). We then test these predictions with quantitative in vivo experiments on intact cells.
Microtubules are biological polymers that alternate between periods of growth and shrinkage. This process, termed dynamic instability by its discovers Mitchison and Kirshner, is crucial for microtubule length regulation, for the exploration of intracellular space, and for cellular force generation. Despite its central role in cell biology, dynamic instability is poorly understood.
We are taking several complementary approaches to understanding dynamics..
(i) We have developed assays to study microtubule dynamics using single-molecule techniques: total-internal reflection fluorescence microscopy (Brouhard et al. 2008), optical tweezers (Jannasch et al. 2013) amd interference reflection microscopy (Mahamdeh et al. 2018).
(ii) We have used these assays to figure out how microtubule-associated proteins (MAPs) regulate microtubule dynamics. We have shown how depolymerizing kinesins target microtubule ends and couple ATP hydrolysis to microtubule shortening (Helenius et al. 2006, Varga et a. 2006, 2009, Friel and Howard 2011); we discovered that XMAP215 is a processive polymerase (Brouhard et al. 2008, Widlund et al. 2011); and we found that the microtubule severing protein spastic also regulates microtubule dynamics to amplify the microtubule cytoskeleton (Kuo et al. 2019).
(iii) We use theory to bridge from single-molecules to macromolecular assemblies (Khataee & Howard 2019, Bowne-Anderson et al. 2013, 2015, Sartori et al. 2016).
Branching morphologenis of neurons
The architecture of the brain and its constituent neurons is staggeringly complex. A fundamental insight into the working of the brain was made by the Spanish anatomist Ramon y Cajal who discovered the “dynamic polarization of neurons” (Ramon y Cajal, Nobel Lecture 1906). According to this principle, neurons are polarized cells, with dendrites and axons, usually on opposite sides of the cell body, and he deduced that the flow of information is also polarized, in a way that respects cell polarity: “nervous movement [i.e. electrical activity] in these prolongations [i.e. the dendrites] is towards the cell or axon, while it is away from the cell in the axons”. By studying the morphology of individual neurons and identifying the dendrites and axons, he was able to map the flow of information through the nervous system. Thus, an insight from cell biology, i.e. cell polarity, provided the key to understanding the functional anatomy of the brain. My intuition is that the cell biological principles underlying branching morphology will likewise provide key information about signaling and information processing by neurons.
Our goal is to understand the principles underlying the branching of neurons, especially dendrites. We and others have shown that the microtubule cytoskeleton play a fundamental role, not just in “feeding” neurons, but also in building them in the first place. Using highly branched mechanoreceptors in flies as a model system, we are trying to understand the tradeoffs between intracellular transport from the cell body into the dendrites, on the one hand, and the flow of information from the dendrites back to the cell body, on the other.
The motility of cilia and flagella
That the motor protein dynein drives microtubule sliding and bending in cilia and flagella has been known since the classic experiments of Gibbons, Brokaw and Satir in the 1960s and 1970s. However, how the activity of the dyneins is coordinated to give a periodic beat remains an open. We are bringing a new constellation of techniques to the problem. In earlier work, we hypothesized that the dyneins are coordinated through a force-sensing mechanism: sliding forces provide positive feedback that switches the activity of the motors across the axis of the axoneme (the motile structure within cilia and flagella) and couples the motors along the length of the axoneme (Riedel-Kruse et al. 2007, Howard 2009). Using high-speed imaging and high-precision image analysis, we showed that our hypothesis can account very satisfactorily for the beat of mammalian sperm.
We are now using the single-celled alga Chlamydomonas Reinhardtii as a model system to study (i) the beating of isolated, reactivated axonemes, the motile structure within cilia and flagella (Geyer et al. 2016, Geyer et al. 2017, Sartori et al. 2016a, Sartori et al. 2016b), (ii) the activity of axonemal subassemblies (Mukundan et al. 2014) and individual dynein molecules purified from these axonemes (Alper et al. 2013, 2014), and (iii) the role of hydrodynamics in shaping the beat pattern (Friedrich et al. 2010) and swimming path (Geyer et al. 2013). In this way we hope to obtain a molecular understanding of one of the most elegant and enigmatic examples of cell locomotion. Oscillators are a paradigms of emergence because the system behavior obscures the contributions of the individual players. We hope to the obtain the first truly molecular and biochemical understanding of a cellular oscillator
We are working on several other projects in which the dynamic and structural properties of microtubules underly important cellular processes such as neuronal polarity, spindle assembly and positioning in mitosis (Coral-Garzon et al. 2016, 2017, Reber et al. 2013, Redemann et al. 2010, Pecreaux et al. 2006, Grill et al. 2003), mechanotransduction (Liang & Howard 2017, Liang et al. 2013, Howard and Bechstedt et al. 2004, Howard and Hudspeth 1988). Also, we are fascinated by the energetics of development (Rodenfels et. al. 2019).
Biophysics; Cilia; Microtubules; Mitosis; Neurobiology; Physics; Developmental Biology; Molecular Motor Proteins; Nanotechnology