Reconstituting microtubule-actin coordination by cytolinkers

The human body is composed of about 3−4×1013 (30-40 trillion) cells [1]. These cells are all functioning consistently, and working elegantly together, to sustain the organism. Not only humans, but all other living things on earth (from plants to parrots) are composed of cells. Cells are the smallest living building blocks of plants and animals, and in fact some organisms are built from a single cell, such as bacteria. To be able to sustain life, cells are dynamic entities that need to grow, divide, and interact with their environment. They accomplish this by a number of complex and dynamic processes. For instance, to perform vital functions such as cell division, a key factor of life, cells need to dramatically change their shape. In addition to shape changes, the internal cellular organization needs to be tightly controlled to properly function. For example, cells need to establish a front-back polarity to drive directional migration, like immune cells that hunt for intruders. Furthermore, the proper functioning of brain cells (also named neurons), which depends on finding and connecting to other neuronal cells, is closely related to their internal organization and cellular shape. For cells, essentially small bags filled with proteins, to change their shape and internal organization, they depend on an internal filamentous scaffold named the cytoskeleton. Unlike the name might suggest, this ’cellular skeleton’ is actually very dynamic, with constant assembly and disassembly of the constituent filaments and changes in filament organization. In addition to organizing the cellular interior, this cytoskeleton provides mechanical support for cells and allows them to generate forces. Two main cytoskeletal components are microtubules and actin filaments. They are usually studied as separate systems, despite a growing body of work indicating their functions are closely intertwined and interdependent. This thesis studies how these two cytoskeletal components influence each other. More specifically, we focus on the question how actin and microtubules co-organize and affect each other via proteins that physically link them to each other, named cytolinkers. To study how cytolinkers impact cytoskeletal crosstalk, we move away from the complex environment of the cell, where many other proteins are present and different processes take place. We took the cytoskeletal building blocks and cytolinking proteins out of the cell, rebuilt a cytoskeleton from these building blocks and characterized the effects of the cytolinkers on cytoskeletal co-organization by fluorescence microscopy. In addition to natural cytolinkers, we engineered our own cytolinkers to better understand how these proteins influence microtubule/actin coordination and in the absence of illdefined regulatory processes in the cell. This isolated context is a powerful tool to study cellular functions, as the simplification allows us to tightly control all variables and identify the underlying mechanisms.BN/Marileen Dogterom LabBN/Gijsje Koenderink La

Revealing the assembly of filamentous proteins with scanning transmission electron microscopy

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filamentsBN/Gijsje Koenderink LabBN/Marileen Dogterom La

A Fully 3D-Printed Steerable Instrument for Minimally Invasive Surgery

In the field of medical instruments, additive manufacturing allows for a drastic reduction in the number of components while improving the functionalities of the final design. In addition, modifications for users’ needs or specific procedures become possible by enabling the production of single customized items. In this work, we present the design of a new fully 3D-printed handheld steerable instrument for laparoscopic surgery, which was mechanically actuated using cables. The pistol-grip handle is based on ergonomic principles and allows for single-hand control of both grasping and omnidirectional steering, while compliant joints and snap-fit connectors enable fast assembly and minimal part count. Additive manufacturing allows for personalization of the handle to each surgeon’s needs by adjusting specific dimensions in the CAD model, which increases the user’s comfort during surgery. Testing showed that the forces on the instrument handle required for steering and grasping were below 15 N, while the grasping force efficiency was calculated to be 10–30%. The instrument combines the advantages of additive manufacturing with regard to personalization and simplified assembly, illustrating a new approach to the design of advanced surgical instruments where the customization for a single procedure or user’s need is a central aspect.Medical Instruments & Bio-Inspired Technolog