Final Report Summary - CELL POLARITY (Role of Microtubule Polarity and Polarized Membrane Traffic in Directed Cell Migration)
Background:
The ability of cells to polarize is crucial for many biological processes, including tissue development, immune response, neurotransmission and wound healing. As many cellular polarity factors play central roles in disease (e.g. cancer, neurological dysfunction) understanding the molecular basis of cell polarity is of great importance to the biomedical sciences. Upon extracellular triggers cells polarize by asymmetrically distributing elements of the cytoskeleton and membrane system, leading to polarized membrane traffic, local cell growth and the establishment of cellular sub-domains. Directed long-range traffic to distinct cellular domains, i.e. the axon of developing neurons or the leading edge of migrating cells, crucially depends on the polarized stabilization of the microtubule (MT) cytoskeleton. Recent studies showed that plasma membrane events, including receptor activation, integrin signaling and recruitment of MT associated factors are necessary for MT stabilization; however, current mechanistic models of how MTs are stabilized remain incomplete. While the function of stable MTs in polarized traffic has been recognized, it has not been investigated, whether local changes in plasma membrane turnover and composition can regulate MT stabilization and polarity. Since both, membrane turnover and MT stabilization, are critical for cell migration, bi-directional functional links between those modules could provide regulatory mechanisms for efficient cell migration.
Objectives
(A) Investigation of the mechanism of Numb/Par3 regulated integrin endocytosis in MT stabilization using cell biological/biochemical approaches.
(B) Investigation of the role of spatiotemporal regulation of MT stabilization factors by employing advanced imaging methods (i.e. live-cell, optogenetics, multi-color TIRF and super-resolution imaging).
(C) Advancement of currently available super-resolution methods to investigate the cytoskeleton and membrane trafficking machinery on the nanoscale.
Results:
Objective A: We used cell biological and high-/super-resolution imaging methods to investigate the molecular mechanism of how microtubule polarity and polarized membrane traffic are linked. In particular, we used an established wound-edge fibroblast system to quantify changes in the level of stable MTs under cell biological manipulations (i.e. knock-down, inhibitors). Our data show novel mechanistic links between integrin endocytosis, phosphoinositide switching and MT stability. Specifically, we observed that two evolutionarily conserved polarity proteins, Numb and Par3, have opposing roles in MT stabilization. The endocytic adaptor Numb acted as a novel suppressor of MT stability. The polarity scaffolding protein Par3, in contrast, was necessary for MT stabilization. We therefore propose an antagonistic regulatory role of Numb and Par3 in MT stability by controlling integrin endocytosis and signaling for MT stabilization. By fine-tuning the amount of stable MTs, Numb/Par3, might comprise important switching modules that regulate the speed or persistency of directed cell migration. We are currently
Objective B: We have used high resolution live-cell time-lapse imaging to investigate the spatiotemporal behavior of Par3 by transient expression of GFP-Par3 in migrating cells. We have identified two distinct pools of Par3, one static pool at cell-cell contacts, as seen previously (Schmoranzer et al., 2009) and one dynamic pool at the leading edge. The dynamic pool consists of vesicular puncta that appear at the leading edge and move towards the cell center, similar to actin retrograde flow. We are currently investigating the functional significance of this dynamic pool in local MT stabilization using cell biological manipulations and advanced imaging methods (i.e. live-cell, optogenetics, multi-color TIRF and super-resolution imaging).
Objective C: Cellular polarity relies crucially on the spatiotemporal organization of sub-cellular proteins and organelles. However, the diffraction limit of conventional microscopy often prevents researchers from resolving sub-cellular structures that are smaller than and closer to each other than 200 nm. Recent technological breakthroughs have given rise to novel fluorescence-based super-resolution imaging methods offering 2 to 10-fold resolution gain over conventional methods. These include structured illumination microscopy (SIM, 2-fold), stimulated emission depletion (STED, 2-6-fold) and single molecule localization methods such as direct stochastic optical reconstruction microscopy (dSTORM, 5-10-fold). Published results using these methods are certainly impressive; however, even after years of development, each of these methods still poses technical challenges regarding their successful application in biology.
The continuing aim of my group is to overcome these limitations to investigate the spatiotemporal distribution and composition of the cellular polarity machinery that are hidden behind the diffraction barrier. Up to date, we have successfully established and applied both SIM and dSTORM methods (Lampe et al., 2012; Lehmann et al., 2015; Podufall et al., 2014; Posor et al., 2013; Sakaba et al., 2013; Watanabe et al., 2014). To reach these goals we systematically addressed the remaining technical challenges in setting up the imaging hardware, optimizing the fluorescent probes and designing acquisition and analysis procedures. Improving the resolution and efficiency of the super-resolution methods will be an ongoing effort in the next years.
Outlook: We will continue using interdisciplinary cell biological and state-of-the-art imaging approaches to investigate the molecular mechanisms of cell polarity. By applying our findings to other polarized cell systems that rely on a stabilized array of MTs, such as developing neurons, we will contribute to a more comprehensive understanding of cell polarity and its associated disorders.
The ability of cells to polarize is crucial for many biological processes, including tissue development, immune response, neurotransmission and wound healing. As many cellular polarity factors play central roles in disease (e.g. cancer, neurological dysfunction) understanding the molecular basis of cell polarity is of great importance to the biomedical sciences. Upon extracellular triggers cells polarize by asymmetrically distributing elements of the cytoskeleton and membrane system, leading to polarized membrane traffic, local cell growth and the establishment of cellular sub-domains. Directed long-range traffic to distinct cellular domains, i.e. the axon of developing neurons or the leading edge of migrating cells, crucially depends on the polarized stabilization of the microtubule (MT) cytoskeleton. Recent studies showed that plasma membrane events, including receptor activation, integrin signaling and recruitment of MT associated factors are necessary for MT stabilization; however, current mechanistic models of how MTs are stabilized remain incomplete. While the function of stable MTs in polarized traffic has been recognized, it has not been investigated, whether local changes in plasma membrane turnover and composition can regulate MT stabilization and polarity. Since both, membrane turnover and MT stabilization, are critical for cell migration, bi-directional functional links between those modules could provide regulatory mechanisms for efficient cell migration.
Objectives
(A) Investigation of the mechanism of Numb/Par3 regulated integrin endocytosis in MT stabilization using cell biological/biochemical approaches.
(B) Investigation of the role of spatiotemporal regulation of MT stabilization factors by employing advanced imaging methods (i.e. live-cell, optogenetics, multi-color TIRF and super-resolution imaging).
(C) Advancement of currently available super-resolution methods to investigate the cytoskeleton and membrane trafficking machinery on the nanoscale.
Results:
Objective A: We used cell biological and high-/super-resolution imaging methods to investigate the molecular mechanism of how microtubule polarity and polarized membrane traffic are linked. In particular, we used an established wound-edge fibroblast system to quantify changes in the level of stable MTs under cell biological manipulations (i.e. knock-down, inhibitors). Our data show novel mechanistic links between integrin endocytosis, phosphoinositide switching and MT stability. Specifically, we observed that two evolutionarily conserved polarity proteins, Numb and Par3, have opposing roles in MT stabilization. The endocytic adaptor Numb acted as a novel suppressor of MT stability. The polarity scaffolding protein Par3, in contrast, was necessary for MT stabilization. We therefore propose an antagonistic regulatory role of Numb and Par3 in MT stability by controlling integrin endocytosis and signaling for MT stabilization. By fine-tuning the amount of stable MTs, Numb/Par3, might comprise important switching modules that regulate the speed or persistency of directed cell migration. We are currently
Objective B: We have used high resolution live-cell time-lapse imaging to investigate the spatiotemporal behavior of Par3 by transient expression of GFP-Par3 in migrating cells. We have identified two distinct pools of Par3, one static pool at cell-cell contacts, as seen previously (Schmoranzer et al., 2009) and one dynamic pool at the leading edge. The dynamic pool consists of vesicular puncta that appear at the leading edge and move towards the cell center, similar to actin retrograde flow. We are currently investigating the functional significance of this dynamic pool in local MT stabilization using cell biological manipulations and advanced imaging methods (i.e. live-cell, optogenetics, multi-color TIRF and super-resolution imaging).
Objective C: Cellular polarity relies crucially on the spatiotemporal organization of sub-cellular proteins and organelles. However, the diffraction limit of conventional microscopy often prevents researchers from resolving sub-cellular structures that are smaller than and closer to each other than 200 nm. Recent technological breakthroughs have given rise to novel fluorescence-based super-resolution imaging methods offering 2 to 10-fold resolution gain over conventional methods. These include structured illumination microscopy (SIM, 2-fold), stimulated emission depletion (STED, 2-6-fold) and single molecule localization methods such as direct stochastic optical reconstruction microscopy (dSTORM, 5-10-fold). Published results using these methods are certainly impressive; however, even after years of development, each of these methods still poses technical challenges regarding their successful application in biology.
The continuing aim of my group is to overcome these limitations to investigate the spatiotemporal distribution and composition of the cellular polarity machinery that are hidden behind the diffraction barrier. Up to date, we have successfully established and applied both SIM and dSTORM methods (Lampe et al., 2012; Lehmann et al., 2015; Podufall et al., 2014; Posor et al., 2013; Sakaba et al., 2013; Watanabe et al., 2014). To reach these goals we systematically addressed the remaining technical challenges in setting up the imaging hardware, optimizing the fluorescent probes and designing acquisition and analysis procedures. Improving the resolution and efficiency of the super-resolution methods will be an ongoing effort in the next years.
Outlook: We will continue using interdisciplinary cell biological and state-of-the-art imaging approaches to investigate the molecular mechanisms of cell polarity. By applying our findings to other polarized cell systems that rely on a stabilized array of MTs, such as developing neurons, we will contribute to a more comprehensive understanding of cell polarity and its associated disorders.