Cells organize membrane-less internal compartments through a process called liquid-liquid phase separation (LLPS) to create chemically distinct compartments, referred to as condensates, which emerge from interactions among biological macromolecules. These condensates include various cytoplasmic structures such as P-granules and stress granules. However, an even wider array of condensates subcompartmentalize the cell nucleus, forming liquid-like structures that range from nucleoli and Cajal bodies to nuclear speckles and gems. Phase separation provides a biophysical assembly mechanism underlying this non-covalent form of fluid compartmentalization and functionalization. In this Cell Science at a Glance article and the accompanying poster, we term these phase-separated liquids that organize the nucleus the liquid nucleome; we discuss examples of biological phase transitions in the nucleus, how the cell utilizes biophysical aspects of phase separation to form and regulate condensates, and suggest interpretations for the role of phase separation in nuclear organization and function.

The liquid nucleome - phase transitions in the nucleus at a glance.

The incorporation of molecular switches in organic structures is of great interest in the chemical design of stimuli-responsive materials that mimic the complex functions of living systems. Merocyanine dyes that convert to spiropyran moieties upon exposure to visible light have been extensively studied as they can be incorporated in hydrated covalent networks that will expel water when this conversion occurs and induce a volumetric shrinkage. We report here on a sulfonate-based water-soluble photoswitch that, in contrast to the well-known systems, triggers a volumetric expansion in hydrogels upon exposure to photons. Contraction is in turn observed under dark conditions in a highly reversible manner. The novel behavior of the photoswitch incorporated in the covalent network was predicted by coarse-grained simulations of the system's chemical structure. Using pH control and polymeric structures that differ in lower critical solution temperature, we were able to develop hydrogels with highly tunable volumetric expansion. The novel molecular function of the systems developed here led to materials with the negative phototaxis observed in plants and could expand the potential use of hydrogels as sensors, soft robots, and actuators.

Light-Driven Expansion of Spiropyran Hydrogels.

Liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and other biomolecules is a highly complex but robust process used by living systems. Drawing inspiration from biology, phase separating proteins have been successfully utilized for promising applications in fields of materials design and drug delivery. These protein-based materials are advantageous due to the ability to finely tune their stimulus-responsive phase behavior and material properties, and the ability to encode biologically active motifs directly into the sequence. The number of possible protein sequences is virtually endless, which makes sequence-based design a rather daunting task, but also attractive due to the amount of control coming from exploration of this variable space. The use of computational methods in this field of research have come to the aid in several aspects, including interpreting experimental results, identifying important structural features and molecular mechanisms capable of explaining the phase behavior, and ultimately providing predictive frameworks for rational design of protein sequences. Here we provide an overview of computational studies focused on phase separating biomolecules and the tools that are available to researchers interested in this topic.

Simulation methods for liquid-liquid phase separation of disordered proteins.

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.

Sensing centromere tension: Aurora B and the regulation of kinetochore function

In the two decades since the introduction of the "click chemistry" concept, the toolbox of "click reactions" has continually expanded, enabling chemists, materials scientists, and biologists to rapidly and selectively build complexity for their applications of interest. Similarly, selective and efficient covalent bond breaking reactions have provided and will continue to provide transformative advances. Here, we review key examples and applications of efficient, selective covalent bond cleavage reactions, which we refer to herein as "clip reactions." The strategic application of clip reactions offers opportunities to tailor the compositions and structures of complex (bio)(macro)molecular systems with exquisite control. Working in concert, click chemistry and clip chemistry offer scientists and engineers powerful methods to address next-generation challenges across the chemical sciences.

Clip Chemistry: Diverse (Bio)(macro)molecular and Material Function through Breaking Covalent Bonds.

Many dynamic biological processes are regulated by protein–protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein–protein interactions.

/pdf/reversible-control-of-protein-localization-in-living-cells-133o3itbym.pdf

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer

To ensure accurate chromosome segregation, interactions between kinetochores and microtubules are regulated by a combination of mechanics and biochemistry. Tension provides a signal to discriminate attachment errors from bi-oriented kinetochores with sisters correctly attached to opposite spindle poles. Biochemically, Aurora B kinase phosphorylates kinetochores to destabilize interactions with microtubules. To link mechanics and biochemistry, current models regard tension as an input signal to locally regulate Aurora B activity. Here, we show that the outcome of kinetochore phosphorylation depends on tension. Using optogenetics to manipulate Aurora B at individual kinetochores, we find that kinase activity promotes microtubule release when tension is high. Conversely, when tension is low, Aurora B activity promotes depolymerization of kinetochore-microtubules while maintaining attachment. Thus, phosphorylation converts a catch-bond, in which tension stabilizes attachments, to a slip-bond, which releases microtubules under tension. We propose that tension is a signal inducing distinct error-correction pathways, with release or depolymerization being advantageous for typical errors characterized by high or low tension, respectively.

Tension promotes kinetochore-microtubule release by Aurora B kinase.

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. By combining the chemical versatility of photoremovable protecting groups with the biological specificity of self-labeling tags, we developed a series of chemi-optogenetic tools that enable protein recruitment with precise spatiotemporal control. To this end, we created a modular platform for chemically inducible proximity (CIP), a technique in which two proteins of interest are brought together by the presence of a small molecule to induce a biological effect. The local proximity of a protein and its substrate has been shown to be sufficient to initiate a desired biological effect, making CIP a valuable technique towards probing cellular processes. The high affinity and specificity of these tags result in rapid initiation of dimerization, allowing biochemical processes to be studied on a biologically relevant timescale. In this chapter, we describe the synthesis and application of chemi-optogenetic probes for spatiotemporal control of protein proximity.

Photoactivatable trimethoprim-based probes for spatiotemporal control of biological processes.

Protein-protein interactions are highly dynamic biological processes that regulate various cellular reactions. They exhibit high specificity and spatiotemporal control in order to efficiently utilize finite resources in a cellular compartment. Photoactivatable chemically inducible dimerization (pCID) has emerged as an attractive technique in the scientific community, leading to the development of systems that can be activated with various wavelengths of light in order to manipulate processes on biologically relevant scales with molecular specificity. These systems can be modified to control various protein functions with unprecedented precision and spatiotemporal resolution. In this chapter, we describe an optogenetic platform that provides reversible control over dimerization of genetically tagged proteins using orthogonal wavelengths of light. We demonstrate photoactivation and photo-reversal of protein localization and transport. Mitosis is manipulated by activating and silencing the spindle assembly checkpoint through recruitment and release of proteins from kinetochores.

Reversible optogenetic control of protein function and localization.

Aurora B kinase regulates kinetochore-microtubule interactions to ensure accurate chromosome segregation in cell division. Tension provides a signal to discriminate attachment errors from bioriented kinetochores with sisters correctly attached to opposite spindle poles. Current models focus on tension as an input to locally regulate Aurora B activity. Here we show that the outcome of Aurora B activity depends on tension. Using an optogenetic approach to manipulate Aurora B at individual kinetochores, we find that kinase activity promotes microtubule release when tension is high. Conversely, when tension is low, Aurora B activity promotes depolymerization of kinetochoremicrotubule bundles while maintaining attachment. We propose that tension is a signal inducing distinct error-correction mechanisms, with release or depolymerization advantageous for typical errors characterized by high or low tension, respectively.

/pdf/tension-promotes-kinetochore-microtubule-release-in-response-4ttrz3nqu7.pdf

Daniel Z Wu

Papers

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer

Tension promotes kinetochore-microtubule release by Aurora B kinase.

Photoactivatable trimethoprim-based probes for spatiotemporal control of biological processes.

Reversible optogenetic control of protein function and localization.

Tension promotes kinetochore-microtubule release in response to Aurora B activity