Ongoing Projects
Mitochondrial transport - mechanisms and function
Regulation of mitochondria-dynein interaction
In our forward genetic screen, we identified a mutant line which shows loss of retrograde mitochondrial transport. This strain has a start site mutation in the gene Actr10 (also known as Arp11). Analysis of the axonal transport of various cargos revealed that only the movement of mitochondria is disrupted in this line. More specifically, only the retrograde (cell body directed) transport of mitochondria is perturbed in the actr10 mutant. We were curious about Actr10’s role in this process. Specifically, we wanted to know if Actr10 was necessary for the physical interaction between mitochondria and the dynein retrograde motor. Using mitochondrial fractionation, we were able to show that Actr10 is essential for mitochondria to bind to the retrograde motor complex, providing insight into the molecular regulation of mitochondria-dynein interaction and subsequent transport in axons. Read more here!
Ongoing work is focused on defining the complete scaffold binding the dynein motor to the organelle. While Actr10 is clearly part of this complex, we are interested in defining the complete proteome of the mitochondria-dynein link.
The function of retrograde mitochondrial transport
While over half of the mitochondrial population is stationary in mature axons, a subset are actively transported. The function of this movement, particularly the retrograde movement of organelles, has been shown to be essential for mitochondrial degradation; however, how disrupting this work impacts the mitochondrial population in the neuron has not been explored. We first addressed the frequency of retrograde transport of mitochondria from axon terminals. Using photoconversion and long-term tracking, we were able to demonstrate that mitochondria turnover from this site within a day. Inhibition of retrograde mitochondrial transport from axon terminals leads to accumulation of unhealthy organelles in the distal axon and, surprisingly, a dramatic loss of cell body mitochondria as well (see below). Together, this indicates that active movement of mitochondria between the cell body and axon terminal is essential for mitochondrial homeostasis in neurons. A preprint describing this work is available. Read more here!
Photoconversion was used to label mitochondria in axon terminals and then these converted (magenta) organelles were tracked over time. This analysis revealed that 50 % of old (magenta) organelles had vacated the axon terminal within a few hours and new (green) organelles had replaced them. (Video by Amrita Mandal)
Mitochondria can be labeled in individual neurons of the lateral line ganglia (magenta above, white below; arrows). This allows for analysis of mitochondrial load in the cell bodies and corresponding axon terminals. When retrograde transport is disrupted in this mutant line, axon terminals accumulate mitochondria at the expense of the cell body. Mitochondrial load - mitochondrial area/area of the neuron analyzed. Scale bars - 10um.
We are interested in expanding on this work to identify the function of retrograde mitochondrial movement in axons. Traveling the entire distance between the axon terminal and cell body is quite a journey. While it is only ~5 mm in zebrafish lateral line axons, similar sensory axons can extend ~ 1 m in humans. Why do these organelles have to move back to the cell body? What is lost when they can’t? We are focused on exploring these questions in zebrafish sensory neurons and cultured mammalian neurons.
Regulation of retrograde cargo transport in axons: The relationship between cargo, motor, and tracks
Motor - cargo attachment
Cargo transport is a highly regulated process. In neurons, cargos must be directed to the correct neuronal compartments for function. Additionally, cargo must be moved and deposited at the right time for use and recycling. How cargos selectively attach to motors is an area of high interest. We are particularly interested in how the single motor protein complex Cytoplasmic dynein can attach to and move a diverse array of cargos to the right place at the right time. Using an unbiased, forward genetic screen, we are identifying modulators of cargo-specific retrograde transport in axons.
Examples of our use of forward genetics in zebrafish to identify regulators of dynein-cargo attachment can be found here and here.
Motor engagement
Several cargos have been shown to bind to anterograde and retrograde motors simultaneously. These cargos include late endosomes, autophagosomes, and mitochondria, among others. This immediately poses the question: How do cargos move unidirectionally, either towards the cell body or axon terminal, when bound to anterograde and retrograde motors at the same time? Work from a number of labs, including ours, indicates that processive, unidirectional transport of cargos bound by anterograde and retrograde motors can be accomplished through regulation of motor activation while they are bound the cargos. This could be through reciprocal regulation be scaffolding proteins, modulators of motor-microtubule binding, and/or regulation of the activity of motor ATPase domains.
Our forward genetic screen identified a mutant line in which cytoplasmic dynein inhibition is lost, altering both axonal transport and regulation of microtubule stability. Join us and our investigation of how this protein works!
In wildtype larvae, axon terminals are finely branched, creating a basket like structure (top left). Acetylated tubulin staining in wildtype (top right) shows linear arrays of stabilized microtubules that enter the terminal. In our novel mutant line (bottom), axon terminals are swollen and contain an expanded network of stabilized microtubules (arrow), a phenotype associated with enhanced dynein activity. (Images by Kate Pinter and Dane Kawano).
Microtubule regulation
Cargo transport in axons is also modulated by the microtubule tracks that the motor walks along. Work to date in vivo and in vitro points to microtubule stability as a regulator of motor function, specifically for the Kinesin family of anterograde motors. We are interested in how microtubule stability is coordinated by Cytoplasmic dynein and how alterations in microtubules affect dynein-mediated transport.
To address these questions, we use in vivo imaging of microtubule dynamics and microtubule stability to directly assess the impact of our genetic modifications on the these microtubule properties in vivo in zebrafish sensory axons.
Imaging microtubule dynamics in vivo in a zebrafish axon. EB3-GFP marks the plus (fast growing) end of microtubules. As microtubules grow, the “comets” can be used to assay distance and velocity of microtubule growth. (Video by Dane Kawano)
Credit to D. Kawano
A forward genetic screen for neuronal cell biology
Zebrafish are an ideal vertebrate for forward genetic screens. They can produce hundreds of progeny from a single breeding pair. Males can be easily mutagenized using alkylating agents. Transgenic strains to mark tissues of interest can be readily made. Larvae that result from matings of these mutated animals can be visualized in large numbers using fluorescent dissecting microscopes to identify mutant lines of interest. Many large screens have been done in zebrafish, which has yielded an array of mutant lines. The first and largest screen has its own issue of Development. You can also read a retrospective by Christiane Nüsslein-Volhard here.
We are conducting a forward genetic screen in zebrafish to identify novel mutant lines with defects specifically in long sensory and motor neuron axons. We are particularly interested in mutant lines with axonal defects consistent with disrupted retrograde transport. For this, we are utilizing a dual transgenic approach to label these two neuron subtypes. To date, we have identified 8 novel mutant lines (4 shown below) with defects consistent with disrupted retrograde transport and are currently looking for new recruits to identify the cellular and molecular mechanisms disrupted that lead to these phenotypes.
Four of the novel mutant lines identified to date. Compared to wildtype (top), all mutants develop normally through larval stages (5 days post-fertilization (dpf) shown). However, axon terminals are swollen (arrowheads; right), a sign of disrupted retrograde transport. To visualize the neurons, GFP is expressed in the neuronal cytoplasm and mitochondria are labeled with mRFP in motor neuron axons.
This is a mid-section of the trunk of a zebrafish larva at 5 dpf. The arrowhead points to an axon terminal of the lateral line sensory axons. Arrows point to motor neuron axons that exit the spinal cord (top).