Cytoskeletal and Actin‐Based Polymerization Motors and Their Role in Minimal Cell Design

Abstract

Life implies motion. In cells, protein‐based active molecular machines drive cell locomotion and intracellular transport, control cell shape, segregate genetic material, and split a cell in two parts. Key players among molecular machines driving these various cell functions are the cytoskeleton and motor proteins that convert chemical bound energy into mechanical work. Findings over the last decades in the field of in vitro reconstitutions of cytoskeletal and motor proteins have elucidated mechanistic details of these active protein systems. For example, a complex spatial and temporal interplay between the cytoskeleton and motor proteins is responsible for the translation of chemically bound energy into (directed) movement and force generation, which eventually governs the emergence of complex cellular functions. Understanding these mechanisms and the design principles of the cytoskeleton and motor proteins builds the basis for mimicking fundamental life processes. Here, a brief overview of actin, prokaryotic actin analogs, and motor proteins and their potential role in the design of a minimal cell from the bottom‐up is provided.

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

“…Out of the different cytomotive filament‐based systems of bacterial plasmid segregation, we used the best‐studied R1/ParMRC machinery . DNA segregation by this system was reconstituted by using parC ‐coated beads that, similar to a previous study, induced ParR‐dependent nucleation of ParM filaments.…”

“…Furthermore, extrachromosomal plasmids possess their own filament‐based DNA segregating systems, typically containing three elements: a centromeric DNA sequence, a DNA‐binding protein, and an ATPase . The most prominent classes of these partitioning systems are type I segregation systems employing ParA‐type ATPases (similar to the chromosome‐segregating systems mentioned above), and type II segregation systems employing actin‐like ATPases of the ParM type .…”

Engineering a Synthetic RNA Segregation System

“…[6][7][8][9] Furthermore, extrachromosomal plasmids possess their own filament-based DNA segregation systems, typically containing three elements: a centromeric DNA sequence, a DNA binding protein, and an ATPase. [10][11][12][13] Most prominent classes of these partitioning systems are type I segregation systems employing ParA-type ATPases (similar to the chromosome segregating systems mentioned above), and type II segregation systems employing actin-like ATPases of the ParM-type. [10] The dynamically unstable ParM polymers consist of polar, left-handed, double-helical filaments and are tethered to the DNA at the growing end via a helical accessory protein complex.…”

Dynamic self-assembly of compartmentalized DNA nanotubes

“…While the cytoskeleton is primarily composed of filaments (including actin and microtubules), its operation is orchestrated by a large number of organizing proteins and interactions with the cell membrane 3 . The development of scaffolding systems that are inspired by the cytoskeleton’s architecture promises to endow synthetic cells and materials with the capacity to adapt, partition, and move 4 – 6 . These scaffolding systems should be easy to customize, and exploit components already established in cell-free synthetic biology.…”

“…The most direct approach to build minimal scaffolds inside artificial cells is that of isolating relevant cellular biomolecules and reconstituting them in cell-sized compartments 5 , 6 . Native cytoskeletal filaments have been encapsulated in a variety of droplets or vesicles; however, achieving dynamic behaviors beyond assembly in confinement is challenging due to both restrictive environmental conditions and laborious purification and reconstitution protocols 7 – 11 .…”