Periodic Reporting for period 4 - GRADIENTSENSING (Cellular navigation along spatial gradients)
Reporting period: 2021-10-01 to 2022-03-31
The global goal of “GradientSensing” is to understand the mechanistic basis of cellular decision making during directed leukocyte locomotion. Directed locomotion in the mammalian context is mainly guided by molecular cues of the chemokine family. These chemokines are often present as gradients, which attract the cells to their source of production. A cell following such gradients does not only need to interpret the distribution of the guidance cue (it has to “see” and follow the gradient). It also has to integrate many other extrinsic factors like the chemical composition of the connective tissue, its geometry and also the cellular composition of the environment.
These questions are essential for the understanding of immune responses as almost all immunological reactions are triggered and executed by cells that are not “on site” but rather get recruited (e.g. from the bloodstream) to the site of action. The mechanistic and molecular understanding of this recruitment process is not only important for our basic biological understanding but any potential (pharmacological) interference can only be built on such mechanistic knowledge. As the project tries to understand very basic and conserved biological features, the relevance reaches far beyond the investigated physiological context. Also during normal development, regeneration and, importantly, during pathological processes like cancer metastasis, cell undergo (mis)guided migration. Hence, the project has potential to guide future therapeutic approaches.
These questions are essential for the understanding of immune responses as almost all immunological reactions are triggered and executed by cells that are not “on site” but rather get recruited (e.g. from the bloodstream) to the site of action. The mechanistic and molecular understanding of this recruitment process is not only important for our basic biological understanding but any potential (pharmacological) interference can only be built on such mechanistic knowledge. As the project tries to understand very basic and conserved biological features, the relevance reaches far beyond the investigated physiological context. Also during normal development, regeneration and, importantly, during pathological processes like cancer metastasis, cell undergo (mis)guided migration. Hence, the project has potential to guide future therapeutic approaches.
We investigated the fundamental question how a migrating cell handles the local geometry of the environment when it “tries” to follow a soluble gradient of chemokine, meaning that the general direction of the path is given by a soluble cue, while the local path is full of physical obstacles. We mechanistically dissected the process of such cellular decision making that has to happen whenever the cell faces two or more conflicting cues. We discovered that the cellular nucleus and the microtubule cytoskeleton both play decisive roles in the coordination of the migratory process and that the nucleus, the most bulky part of the cell, essentially guides the cell along the “path of least resistance”. The resulting publication (Renkawitz et al, Nature, 2019) was the base of a video the journal Nature produced on cell migration. The video is very illustrative and reached a broader public audience:
https://www.youtube.com/watch?v=uKid8QmBI0I&t=15s(opens in new window)
This migration along the path of least resistance creates a challenge that comes along with the necessity to decide which of multiple competing cellular protrusions “wins” over the others and so guides the cell along its path. In order to coordinate this process the cell has to sense its own shape and take care that coherence of the cytoplasm is always maintained. If this principle fails the cell will tear itself into pieces and fragment into multiple cytoplasts. We identified a molecular pathway of “cellular proprioception” that guarantees that only one chemotactic protrusion dominates and all others are retracted. If this pathway is eliminated, chemotactic cells entangle in complex 3D environments and dissociate into multiple (still migratory) cytoplasts and ultimately die. At the core of this pathway is the microtubule cytoskeleton and we show how microtubules serve to “measure” the inner space of the cell. This work was published in J Cell Biol (Kopf et al, J Cell Biol, 2020).
When moving up a chemotactic gradient, cells do not only face physical obstacles (see above) but they also encounter different chemical environments, meaning that the surfaces they encounter (usually in the form of other cells or extracellular matrix) have adhesive features. For a cell following a soluble gradient these features should be of secondary relevance as the guidance cue always serves as the dominant signal. Accordingly (as we showed previously) substrate adhesions are largely dispensable for chemotactic leukocytes. Which principle the cells use instead was not known. In a reductionist proof of principle study we could demonstrate that it is the amoeboid (shape changing) principle alone that can propel the cells, provided that they are in a geometrically sufficiently complex environment. We provide a detailed theoretical and experimental framework to explain how the actin cytoskeleton achieves this task. The paper was published in Nature (Reversat et al, Nature, 2020).
https://www.youtube.com/watch?v=uKid8QmBI0I&t=15s(opens in new window)
This migration along the path of least resistance creates a challenge that comes along with the necessity to decide which of multiple competing cellular protrusions “wins” over the others and so guides the cell along its path. In order to coordinate this process the cell has to sense its own shape and take care that coherence of the cytoplasm is always maintained. If this principle fails the cell will tear itself into pieces and fragment into multiple cytoplasts. We identified a molecular pathway of “cellular proprioception” that guarantees that only one chemotactic protrusion dominates and all others are retracted. If this pathway is eliminated, chemotactic cells entangle in complex 3D environments and dissociate into multiple (still migratory) cytoplasts and ultimately die. At the core of this pathway is the microtubule cytoskeleton and we show how microtubules serve to “measure” the inner space of the cell. This work was published in J Cell Biol (Kopf et al, J Cell Biol, 2020).
When moving up a chemotactic gradient, cells do not only face physical obstacles (see above) but they also encounter different chemical environments, meaning that the surfaces they encounter (usually in the form of other cells or extracellular matrix) have adhesive features. For a cell following a soluble gradient these features should be of secondary relevance as the guidance cue always serves as the dominant signal. Accordingly (as we showed previously) substrate adhesions are largely dispensable for chemotactic leukocytes. Which principle the cells use instead was not known. In a reductionist proof of principle study we could demonstrate that it is the amoeboid (shape changing) principle alone that can propel the cells, provided that they are in a geometrically sufficiently complex environment. We provide a detailed theoretical and experimental framework to explain how the actin cytoskeleton achieves this task. The paper was published in Nature (Reversat et al, Nature, 2020).
Beyond these published studies we have exciting results regarding the actual gradient sensing process of chemotaxing leukocytes. To our big surprise we found that in one of our model systems (Dendritic cells) the actual formation of gradients is not as straight-forward as we previously anticipated. The prevailing paradigm that we followed was that these chemokine gradients are generated by interstitial diffusion and subsequent immobilization to sugars in the interstitium. We found that there is a step involved where the cells that actually sense the gradient substantially change the gradient before they sense it. This means that the chemotactic cells manipulate the gradient while they move along it. We consider this a highly interesting self-organizing feature with broad biological relevance and currently try to understand its implications in the inflammatory context. We developed a number of novel microfluidic devices as well as more physiological (cell derived) matrices to show how this principle of self generating gradients allows the cells to gain collective features to migrate more efficiently up preformed gradients. We are close to finishing a first publication on this topic. While we are highly excited about the conceptual biological relevance of this finding, it also forced us to re-consider many of our findings in the field of actual gradient-perception. Here, we always assumed that the gradient is prescribed externally. The finding that the cells change the gradient made it necessary to refine our methodology in this field.
Finally, we made significant progress on the technology-development side (work package 3) and managed to optimize a genome-editing protocol that allows for the first time to do homozygous knock-in mutants in HoxB8 hematopoietic precursor cells.
Finally, we made significant progress on the technology-development side (work package 3) and managed to optimize a genome-editing protocol that allows for the first time to do homozygous knock-in mutants in HoxB8 hematopoietic precursor cells.