Jeffrey van Haren received his PhD in cell biology from Erasmus MC, where he studied the regulation of the mammalian microtubule cytoskeleton. He did his postdoctoral training in the Wittmann lab at UCSF, where he developed novel tools to control microtubule dynamics with light. In 2019 he started his own research group at Erasmus MC. His work uses Mightex’s Polygon Pattern Illuminator.

Spatial Control of Microtubule Dynamics at the Subcellular Scale by Patterned Illumination(Application Note, 2018)

Overview

Our lab focuses on elucidating the molecular mechanisms that control the organization of the mammalian microtubule (MT) cytoskeleton, a network of highly dynamic tube-shaped polymers inside all of our cells that facilitates intracellular transport, controls chromosome segregation, serves to organize the cell interior, and allows cells to establish and maintain polarity. Proper organization of MTs inside cells requires precise spatial and temporal control of MT assembly/disassembly, but it has been difficult to study how MT regulatory factors accomplish such precise local control.

Methods and Results

To gain a better understanding of these mechanisms, we have developed novel optogenetic tools that enable precise spatiotemporal control of the microtubule cytoskeleton with blue light. In collaboration with the Hahn lab at UNC, we devised a strategy to optically control proteins by introducing a small light-sensitive protein pair into a protein of interest. We used this strategy to develop a Photo-Inactivated variant of the key microtubule regulator EB1 (termed π-EB1), and generated cells in which π-EB1 replaced the wildtype protein (see Figure 1A and B). Illuminating these π-EB1 cells with blue light caused the nearly instant inactivation of π-EB1. Due to the rapid reversibility of this system, turning off the blue light resulted in rapid re-activation of π-EB1 and thereby allowed us to spatially control π-EB1 in small regions of the cell using the Mightex Polygon pattern illuminator (see Figure 1C and D). This in turn allowed us to spatially control microtubule dynamics within cells and to study the immediate consequences of locally altering MT growth dynamics. Local inactivation of π-EB1 in migrating cells caused cells to directly turn away from the area of π-EB1 inactivation. A manuscript describing our findings was recently published (van Haren et al. 2018, Nature Cell Biology).

Figure 1. (A) Schematic of the photo-inactivated π-EB1 design resulting in the reversible dissociation of the MT binding and +TIP adapter domains of EB1 upon blue light exposure. (B) Cell expressing mCherry-tubulin (microtubules), GFP-Zdk1-EB1C and unlabeled EB1N-LOV2 before and after blue light induced π-EB1 dissociation. Yellow arrowheads mark two microtubule ends before and after π-EB1 inactivation, and clearly show the loss of GFP-Zdk1-EB1C from microtubule ends after blue light illumination. (C) Color-coded maximum intensity projection of a time-lapse recording of cells expressing π-EB1. The lower half of the field of view was exposed to blue light using a digital micromirror device (Polygon, Mightex). Note the absence of EB1 comet tracks in the illuminated area, indicating that π-EB1 was locally inactivated. (D) π-EB1 cell expressing mCherry-tubulin and constitutively active Rac1, before and after π-EB1 inactivation in the right half of the cell (blue box marks area illuminated by the Polygon). Yellow arrowheads mark microtubules close to the cell edge (red lines mark a ~7.5 µm wide zone at the cell edge). Note the striking reduction of MTs at the cell edge after blue light inactivation (t = 180 s) of π-EB1 (right panel).

Future Directions

This technique is likely applicable to many other proteins, and will be of great value to gain a better understanding of the mechanisms that control cell polarization. Current work is focused on elucidating local functions of EB1 during neuronal development.

Local Control of Intracellular Microtubule Dynamics by EB1 Photodissociation(Nature Cell Biology, 2018)

Overview

Cellular architecture and behaviour is largely dependent on the organization and dynamics of the microtubule cytoskeleton. Coordination of microtubules inside cells is regulated by diverse microtubule-binding proteins, which in turn recruit effector proteins that control microtubule growth. A recently published paper in Nature Cell Biology from Torsten Wittmann’s lab at UCSF illustrated the development of a novel optogenetic system to manipulate microtubule organization. Jeffrey van Haren et al. used targeted light stimulation to precisely inactivate the microtubule-binding protein End-binding protein 1 (EB1), which is responsible for binding microtubules and recruiting plus-end-tracking proteins (+TIPs) to their positive ends.

Methods

Targeted inactivation of EB1 was achieved by employing Mightex’s DMD-based Polygon pattern illuminator, which allowed the researchers to illuminate multiple, discrete, high-contrast patterns at the same time. In this paper, the Polygon was coupled to the spare camera port on the microscope to enable simultaneous imaging (spinning disk) and targeted photostimulation (Polygon). Select areas of the cell were illuminated using the Polygon and illumination was synchronized with the camera via TTL pulses, such that the camera would trigger 10–20 ms pulses of blue light from the Polygon in between image acquisitions. The intermittent short pulses of blue light stimulation removed any scattered photostimulation light from the imaging.

Results

The researchers were able to locally induce photodissociation of EB1 within cells and disrupt microtubule organization in those regions of interest. In one particularly elegant experiment, Jeffrey van Haren et al. showed that local EB1-dissociation-based microtubule disruption led to a change in orientation of the migrating leading edge of cancer cells. With the Polygon, the researchers were able to generate an optical cage, such that cancer cells within a projected blue-light boundary would change direction once they reached the blue light and, thus, they would be trapped within the optical cage. These findings implicated the role of EB1 interaction with +TIPs in maintaining the directionality of migrating cells.

Jeffrey van Haren et al. show how the development of optogenetic probes for cell biology combined with the Polygon DMD-based technology opens the door for new intriguing findings in the cell biology field.

Reference

van Haren, J., Charafeddine, R.A., Ettinger, A., Wang, H., Hahn, K.M., & Wittmann, T. (2018). Local control of intracellular microtubule dynamics by EB1 photodissociation. Nature Cell Biology.