Chemical tools for dissecting cell division

Abstract

Components of the cell division machinery typically function at varying cell cycle stages and intracellular locations. To dissect cellular mechanisms during the rapid division process, small-molecule probes act as complementary approaches to genetic manipulations, with advantages of temporal and in some cases spatial control and applicability to multiple model systems. This Review focuses on recent advances in chemical probes and applications to address select questions in cell division. We discuss uses of both enzyme inhibitors and chemical inducers of dimerization, as well as emerging techniques to promote future investigations. Overall, these concepts may open new research directions for applying chemical probes to advance cell biology.

Figure 1.. Chemically induced dimerization recruits effector proteins to anchors.

(a) A small-molecule dimerizer binds to a pair of protein targets, each of which is fused to an effector or anchor protein. If the anchor is localized to an intracellular structure, dimerizer addition recruits the effector to that structure. (b) TMP-Halo dimerizer. The bifunctional linker includes a TMP moiety and a Halo ligand to dimerize proteins fused to an Escherichia coli dihydrofoliate reductase (eDHFR) and a Halo enzyme. (c) Schematic of light-induced dimerization using CTH. A coumarin-caged dimerizer initially binds to the Halo-tagged anchor protein. Upon light-uncaging, the exposed TMP moiety recruits eDHFR-tagged effectors to anchors. (d) Schematic of light-induced cleavage using TNH. The photo-cleavable moiety, NVOC, inserted between TMP and Halo, releases the bound effectors from anchors upon light activation.

Figure 2.. Centrinone inhibits PLK4 activity.

(a) Mitotic spindle formation. After nuclear envelope breakdown (dashed lines), centrosomes act as microtubule organizing centers to shape the spindle. Dynamic spindle microtubules capture chromosomes for later congression and segregation. (b) Centrinone prevents centriole duplication by inhibition of PLK4. Because PLK4 has dual activities of auto-activation and phospho-regulated proteolysis, centrinone washout induces a wave of PLK4 activity that generates over-duplicated centrioles in cancer cells. After several cell cycles, the centriole number eventually returns to the initial “set point”. (c) Centrinone generates asymmetric spindles. By blocking centriole duplication in S phase, the cell contains halved centriole numbers in mitosis (1:1 spindle). In the next cycle, the daughter cells contain only one isolated centriole (1:0 spindle), resulting in asymmetric spindles.

Figure 3.. Kinesin motors on spindle microtubules.

(a) Chromosome congression upon establishing spindle bipolarity. Eg5 (kinesin-5) and KIF15 (kinesin-12) motors cross-link and powerstroke on microtubule arrays to establish spindle bipolarity. CENP-E (kinesin-7) motors translocate uncongressed chromosomes from spindle poles to the equator. (b) Inhibition modes of kinesin motors. Rigor inhibition traps kinesin in a strong-binding state on microtubules, whereas inhibitors inducing a weak-binding state or genetic knockdown decrease the number of microtubule-bound motors. (c) K-fibers are mechanically coupled in the spindle. Kinesin motors cross-link interpolar microtubules, k-fibers, and bridging fibers. (d) Centromere relaxation assay. Centromeres are stretched by k-fibers pulling bi-oriented sister kinetochores in opposite directions, and relax immediately following k-fiber ablation a few microns away from one kinetochore. Strong relaxation indicates weak load-bearing on k-fibers. (e) Chromosome congression assay. CENP-E recruitment to kinetochores of pole-proximal chromosomes drives movement towards the metaphase plate, whereas kinesin-1 transports chromosomes away from the pole in all directions.

Figure 4.. Aurora kinases regulate kinetochore-microtubule interactions.

(a) Lack of tension across a sister kinetochore pair provides a signal to identify incorrect attachments, which are destabilized to allow new attachments to form. This process continues until all attachment errors are resolved. (b-c) Effects of recruiting Aurora B to kinetochores to increase local kinase activity. On monopolar spindles (b), kinetochore recruitment primarily triggers k-fiber depolymerization to move the chromosome toward the attached pole. Conversely, recruiting Aurora B to a single kinetochore of a bi-oriented sister pair (c) primarily induces k-fiber release, with the targeted kinetochore moving away from the pole that it was initially attached to. (d) Aurora A localizes to spindle poles and phosphorylates kinetochores near the pole to destabilize microtubule attachments.

Figure 5.. Activating and silencing the SAC.

(a) The SAC is activated by unattached kinetochores to delay anaphase onset until all chromosomes are attached to spindle microtubules (SAC silencing). (b) End-on microtubule attachment at kinetochores silences the SAC. Microtubule-bound DAM1 complex blocks the interaction between MPS1 at the outer kinetochore and SPC105 at the inner-kinetochore. (c) SAC reactivation by rapamycin-induced dimerization. Recruiting exogenous MPS1 close to SPC105 or recruiting a domain of SPC105 close to MPS1 is sufficient to reactivate the SAC.

Figure 6.. Prospective strategies for modulating enzymatic activities.

(a) Inducible protein degradation. Auxin/IAA addition recruits AID-tagged target proteins to the AID-recognition component SCF^TIR1, which catalyzes poly-ubiquitination to degrade the target. (b) Photo-sensitive degron. Light triggers a conformational change in the LOV2 domain, which exposes the degron to recruit E3 ligases and degrade the target. (c) Recognition-inhibition strategy. A low-affinity inhibitory protein displaces the original high-affinity substrate by increasing its local concentration by recruitment to the target. Possible candidates for inhibitory proteins include user-designed nanobodies or monobodies. (d) Local kinase inhibition (LoKI) strategy. Kinase inhibitors conjugated with the SNAP-tag ligand CLP can be locally concentrated proximal to SNAP-tagged proteins. In this example, by fusing a SNAP tag to the centrosome-localized PACT domain, inhibitors are concentrated at spindle poles. (e) Droplet-assisted recruitment. A peptide induces droplet formation upon surpassing its critical concentration, thereby increasing the recruitment of effectors. (f) Hook effect of chemically induced dimerization. Scenarios of limiting or excessive dimerizers reduce dimerization efficacy.