Paul Forscher - US grants

During the pathfinding phase of neuronal development, the growth cone functions as a specialized sensor, capable of guiding extending axons toward distant target sites. Growth cones display a high level of actin-based motility which, in an as yet unspecified manner, supports this guidance process. Specific receptors on the growth cone surface also appear to be involved in growth cone guidance. Some of these receptors interact with components of the extracellular matrix that may serve as spatial cues. Although there has been intense interest in characterizing molecules involved in neuronal guidance, very little is known about the actual signal transduction processes involved; for example, how receptor occupation leads to dynamic alterations of cytoskeletal structure and motility likely to underlie pathfinding decisions. The proposed research attempts to fill this gap in our knowledge: (1) by characterizing signal transduction mechanisms involved in regulation of growth cone motility and structure and (2) by investigating the basic mechanochemical processes that sustain this motility. The results of this work should have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and may also exceed our understanding of the process of nerve regeneration. Diagnostic probes for molecular substrates involved in developmental and regenerative neuronal disorders could also result from the proposed research. A model system employing high resolution digital-video imaging of microscopic membrane surface probes in living neurons will be used to investigate signal transduction mechanisms involved in growth cone guidance. Specific probes for receptors thought to be involved in the processes of guidance and target recognition will be developed. These membrane probes will then be used to investigate alterations of growth cone cytoskeletal structure and motility that appear to occur in response to appropriate extracellular guidance cues. The ultimate goal of the signal transduction section of this project is to characterize in detail receptor mediated processes that regulate membrane-cytoskeletal interactions and actomyosin-based motility in growth cones and relate these molecular events to the macroscopic problem of growth cone guidance. How growth cones generate locomotive force is not known; thus, the second phase of this proposal involves characterization of the mechanochemical processes and cytoskeletal dynamics underlying growth cone motility. A clearer understanding of growth cone mechanochemistry will compliment the signal transduction questions outlined above. The identity, spatial localization and biochemical properties of neuronal myosin-like molecules involved in actin-based movements in growth cones will be investigated. Neuronal actin-myosin interactions will be approached by attempting to reconstitute neuronal actin-myosin motility in vitro in a demembranated cell model system. Further characterization of actin dynamics in intact growth cones will also be undertaken using fluorescent analog cytochemistry.

During the pathfinding phase of neuronal development, the growth cone functions as a specialized sensor, capable of guiding extending axons toward distant target sites. Growth cones display a high level of actin- based motility which, in an as yet unspecified manner, supports this guidance process. Specific receptors on the growth cone surface also appear to be involved: some of these receptors interact with extracellular matrix components that may serve as spatial cues, still others recognize cell adhesion molecules (CAMs) on other cell surfaces. Although there has been intense interest in characterizing molecules involved in neuronal guidance, very little is known about the actual signal transduction processes involved; for example, how receptor occupation leads to alterations of cytoskeletal structure and motility likely to underlie pathfinding decisions. The proposed research attempts to fill this gap in our knowledge: (1) by characterizing signal transduction mechanisms involved in regulation of growth cone motility and structure and (2) by investigating the molecular dynamics underlying the motility process itself. The results of this work have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and will extend our understanding of the process of nerve regeneration. Diagnostic probes for developmental and regenerative neuronal disorders could also result from the proposed research. This project relies on the use of high spatial and temporal resolution digital imaging techniques to investigate the behavior of membrane proteins involved in growth cone guidance and synaptogenesis. To achieve these ends, a system utilizing pseudosubstrate probes has been developed that allows tracking of membrane proteins in growth cones. These probes (typically 100-200 nm beads derivatized with ligands of interest) are being used to investigate alterations of growth cone cytoskeletal structure and motility that occur both in response to the pseudosubstrates themselves and during growth cone target interactions. To facilitate the pseudosubstrate experiments, a single beam gradient optical trap (laser tweezers) will be constructed. The laser tweezers is a non invasive method for micropositioning of small objects (like microbeads) which can also be used to measure forces associated with the growth cone guidance and recognition processes. To compliment these studies, intracellular actin dynamics will be characterized using fluorescence photoactivation techniques. This will allow us to compare and contrast intracellular f-actin and cell surface protein movements involved in growth cone target recognition or substrate adhesion. Finally, a reverse genetic approach will be used to generate specific molecular probes for proteins likely to be involved in regulation of growth cone motility. Specifically, fusion proteins immunogens expressed subsequent to gene cloning from an Aplysia cDNA library will be used to generate antibodies to CAM and actin binding proteins of interest.

[unreadable] DESCRIPTION (provided by applicant): Rho GTPases are molecular switches that play well characterized roles in cell adhesion, polarity, and control of cytoskeletal protein dynamics in non-neuronal cells. Although functionally implicated in axon guidance and nerve regeneration, neuronal Rho GTPase cell biology is far less well understood. To address this gap, we recently used multi-mode Fluorescent Speckle Microscopy (FSM), which allows direct measurement of cytoskeletal protein dynamics, to investigate Rho dependent responses evoked by the chemorepellant agent, lysophosphatidic acid (LPA). We discovered that LPA treatment increased the contractility of "actin arc" and actin bundle structures within the growth cone and provided evidence that this contractility was driving Rho/Rho Kinase dependent growth cone retractions. Actin arc contractility appears to involve Myosin II since it depends on Myosin Light Chain Kinase and Myosin Light Chain phosphatase activities. Other work suggests Rho GTPase and Ca activity can modulate the polarity of axon guidance responses to a single ligand. Although these findings have caught the attention of axon guidance and nerve regeneration fields, the cell biological mechanisms underlying response switching are poorly understood. We have preliminary evidence that increasing background Rac activity converts LPA retraction responses to neurite growth and advance. Interestingly, such LPA evoked growth is accompanied by increases in intracellular Ca and loss of actin arc contractility -i.e. the opposite of what is observed without Rac activation. We propose to combine use of Fluorescent Speckle Microscopy and Ca Imaging to quantitatively assess actin, microtubule, and Calcium dynamics in growth cones to investigate the cytoskeletal and signaling mechanisms underlying apparent switching of LPA response polarity by Rac activity. We will also characterize the role of Myosin II in these responses and do correlative ultrastructural studies to better define the cell biology of this important molecular motor in the growth cone. Our working hypothesis is that Rac and Rho GTPases can modulate the functional output of ligand activated responses via specific effects on MT dynamics which in turn affect localization of ER Ca stores and regulate the Ca release topography and/or release sensitivity. This hypothesis will be tested in the context of LPA as well as Ephrin and Slit ligands, the latter two being known to exert their chemorepellant responses via activation of Rho in the CNS. [unreadable] [unreadable]

This is an award to develop Holographic Optical Tweezers (HOT) into a instrument for quantitative investigation of mechanotransduction in cells. This instrument will be uniquely capable of quantifying the structure and dynamics of the cytoskeleton with fluorescent speckle microscopy (FSM) while biochemical signals and mechanical forces are simultaneously controlled with HOT. A spinning-disc confocal microscope will provide images of the cytoskeleton for FSM, while a programmable closed-loop HOT system will simultaneously manipulate up to about one hundred colloidal particles in 4-D. These particles will be functionalized with a number of different biochemical molecules and will be attached to the cell membrane in any desired pattern. Simultaneous trapping of multiple beads will enable the application of nanonewton level forces to single cells - an increase of up to two orders of magnitude over existing optical methods. The closed-loop control system will be capable of generating large forces and, with the help of novel low-level feature recognition and hologram calculation codes, it will also be capable of controlling and measuring forces at multiple locations in real time - with resolutions as fine as 10 fN at 30Hz.

The investigators will work closely with academic and industrial partners to ensure that the proposed technology is widely adopted. This interdisciplinary project will bring together students and post-docs from engineering and biology. Through the course of the proposed research, they will develop a broad set of skills in colloid science, optics, molecular biology and cell biology. This interdisciplinary training will prepare them for innovative careers in nanoscience and nanotechnology at the intersection of the physical and biological sciences. Furthermore, the proposed instrument will be employed in an advanced course in optical microscopy for graduate students in the physical and biological sciences. The investigators will continue to encourage members of underrepresented groups to contribute to their research.

DESCRIPTION (provided by applicant): Neurons are the most polarized cells known. Regulated assembly and disassembly of actin filament and microtubule cytoskeletal proteins is fundamental to generation of neuronal polarity and regulation of neuronal growth. Recent developments in imaging technology including fluorescent speckle microscopy (FSM) enable direct visualization of cytoskeletal polymer assembly, disassembly and translocation of in living cells. New algorithms for automated fluorescent feature tracking allow quantitative analysis of protein movements on an image-wide basis, allowing characterization of cytoskeletal system responses. These computational advances also pave the way for mathematical modeling of neuronal motility events. We have been using these techniques to characterize the actin filament and microtubule protein dynamics involved in neuronal growth. Our focus has been on the highly motile structure located at the distal end of developing axonal and dendritic processes called the growth cone. Growth cones regulate the rate and direction of neurite advance by responding to diffusible and/or bound molecular cues in their environment. Cue recognition feeds into signal transduction pathways that ultimately impinge on the final cytoskeletal protein effectors. Retrograde actin flow is a ubiquitous aspect of growth cone motility. It results from constant actin network assembly at the leading edge plus rearward network pulling due to myosin II contractility in a more central region of the growth cone called the T zone. Much recent work has focused on proteins that coordinate the actin filament assembly at the leading edge necessary to sustain retrograde flow. However, actin filament turnover is also necessary to maintain the steady state polymer flux characteristic of retrograde flow. This project is aimed at addressing the problem of actin turnover and network recycling in neuronal growth cones since this important function is not understood. We are motivated by our recent discovery that localized myosin II contractility plays an unexpected role in the steady turnover of polarized actin bundles that comprise filopodia. These actin bundles are assembled at the leading edge, and then transported by retrograde flow into the T zone where they undergo minus end severing and turnover. Our studies suggest that localized myosin II contractility potentiates the activity of a separate actin filament severing factor. We propose to investigate cofilin as an actin bundle recycling candidate and characterize cofilin function elsewhere as well. Although cofilin has been implicated in growth cone function, its mechanism of action in cells is not well understood; indeed, recent biochemical evidence suggests cofilin actions are complex and concentration dependent. To address this outstanding problem, novel molecular imaging assays will be used to correlate actin filament turnover rates directly with actin filament-cofilin interactions in living growth cones. We will also look at cofilin function in the context of acute growth cone advance stimulated by application of permissive cell adhesion molecule substrates. PUBLIC HEALTH RELEVANCE: Neuronal growth cone motility is necessary for axon growth and guidance during development and for regeneration after nerve injury. Growth cone motility depends on the steady assembly and disassembly of actin filament networks. How and where the relevant actin networks assemble is relatively well understood; however, the network disassembly process is a mystery. This project addresses this outstanding problem.

DESCRIPTION (provided by applicant): The ADF/Cofilin family of proteins plays a critical role in actin filament turnover essential to all forms of eukaryotic cell motility and normal brain development. Despite a vast literature on signaling pathways controlling cofilin activity, assessing cofilin function in living cells has been hampered by lack of real time assays of cofilin activity. In Aim I will take advantage of the intrinsic ATPase activity of actin filaments and high affinity of active cofilin for ADP-actin subunits to implement an assay for cofilin activity and dynamics in living neurons using quantitative fluorescent speckle microscopy (qFSM). Aplysia cofilins will be derivatized at specific sites with AlexaFluor tags and their biochemical activity and functionality verified in vitro. AlexaFluor-apCofilins and AlexaFluor-G-actin will then be injected into Aplysia neurons and low levels for qFSM and speckle dynamics recorded under conditions of varying apCofilin activity. apCofilin vs actin speckle kinematics, speckle lifetimes, and turnover dynamics will be analyzed. Effects on cofilin activity will be correlated with actin filament structure assessed by light and electron microscopy. Myosin II dependent mechanical forces have been reported to affect cofilin severing activity; thus, we will investigate whether Myosin II activity directly affect cofilin activity and actin dynamics during neurite outgrowth. Ths robust cofilin activity assay is portable to other cells types and will provide a valuable new tool for addressing regulation of cell motility processes including axon growth and regeneration. Neurite outgrowth is characterized by coordinated advance of the central (C) and peripheral (P) cytoplasmic growth cone domains. We recently reported that serotonin (5-HT) accelerates rates of neurite outgrowth by ~300% via a mechanism involving phospholipase C (PLC) dependent Ca release and calcineurin (CN) dependent activation of cofilin in the growth cone P domain. 5-HT stimulated outgrowth was accompanied by CN dependent increases in retrograde actin filament flow in the P domain. When background non-muscle Myosin II activity was inhibited, 5-HT continued to trigger cofilin activation and increases in retrograde actin flow but C domain advance no longer occurred. Thus, myosin II activity is necessary for functionally coupling increases in actin treadmilling in the P domain with advance of the C domain. In Aim II-III we address why this is so. We have previously implicated Rho kinase (ROCK) in regulation of Myosin II dependent C domain contractility and will investigate a role for ROCK in coordination of C and P domain function. Experiments are proposed to generalize the cytoskeletal mechanisms being studied to the many other growth factor receptors that utilize PLC signaling. These studies are predicted to have significant clinical implications for understanding neurodegenerative disease and nerve regeneration related to brain and/or spinal cord injury.

The ADF/cofilin family of proteins play a critical role in actin filament turnover essential to all forms of eukaryotic cell motility. Despite a vast literature on signaling pathways controlling cofilin activity, assessing the function of this important protein in living cells has been hampered by lack of real time assays of cofilin function. Using a novel real time assay for assessing cofilin activity and actin dynamics simultaneously by quantitative fluorescent speckle microscopy (qFSM), we have discovered that mechanical stress imposed on treadmilling actin networks increases cofilin activity with dramatic effects on actin turnover rates that depend on the level of stress. Low stress levels are associated with increases in actin turnover and treadmilling rates that are associated with chemotropic growth; in contrast, stress levels above a critical threshold lead to catastrophic decreases in actin network density resulting in neurite retraction. We have been studying mechanical effects on cofilin activity in the context of serotonin (5-HT) evoked neurite growth responses mediated by classical G(q) subtype GPCRs, which activate phospholipase C to generate IP3 and DAG signals. 5-HT evokes IP3 dependent Ca release from intracellular stores and cofilin activation by a Ca?calcineurin signaling cascade. We now have evidence that DAG production results in PKC dependent increases in non-muscle myosin II activity. This in turn generates local network stress and mechano-catalytic activation of cofilin resulting in local alteration of F-actin structure and network turnover rates. Effects on actin structure also depend on the level of PKC activation. PKC has other known roles including regulation of microtubule (MT) dynamics in growth cones and since MTs are the transport substrate for ER/Ca stores, MT dynamics regulate the functional topography of IP3 dependent Ca release involved in 5-HT dependent growth. PKC can also potentiate integrin based cell adhesion and thereby affect traction forces involved in neuronal growth. We propose to investigate PKC as a signaling node that coordinates: 1) myosin II contractility, 2) actin turnover via cofilin mechano-catalysis, 3) Ca release topography via regulation of microtubule/ER dynamics, and 4) ultimately, traction force production during axon growth. These studies will provide a mechanistic framework for understanding how cofilin enables functional crosstalk between actin dynamics and myosin II contractility during chemotropic growth responses. The results will have interesting implications regarding the key role PKC plays in neuronal growth and neurodegenerative disease.