Casper Hoogenraad

Genentech; South San Francisco CA, United States of America

He, Liu et al. (2021) International Worm Meeting "PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) redundantly mediate MTOC localization and microtubule polarity in dendrites"

Hoogenraad, Casper, He, Liu, Harterink, Martin

[International Worm Meeting, 2021]

In neurons the microtubule cytoskeleton is key for proper axon and dendrite deployment. In axons microtubules are arranged with their plus ends distal to the cell body, whereas dendritic microtubules are predominantly arranged with their minus ends distal to the cell body (invertebrates) or with mixed orientation (vertebrates). This difference in microtubule organization allows for selective transport into axons or dendrites to differentiate these cell extensions. It was recently found in C. elegans that during dendrite development of the PVD neuron, RAB-11 positive vesicles function as a non-centrosomal microtubule organizing center (MTOC). These vesicles localize to the growing dendritic tip to nucleate microtubules with their characteristic minus ends distal organization (Liang et al. eLife, 2020). However, the mechanism that localizes these MTOC vesicles to the dendrite tip is poorly understood. Looking for proteins that may act at the microtubule minus ends, we found that PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) act redundantly for distal MTOC localization during dendrite development; their loss of function leads to defects in microtubule polarity establishment. We found that NOCA-2 colocalizes to the MTOC vesicles, and its depletion partially disrupted the recruitment of gamma-Tubulin to the MTOC vesicles. CAMSAP proteins are well described microtubule minus-end binding proteins that can stabilize/protect the minus-end from depolymerization. Not surprisingly we observed a punctate PTRN-1 distribution throughout the dendrite but also an accumulation around the MTOC vesicles. Surprisingly we found that during dendrite tip growth the MTOC vesicles and the surrounding Camsap puncta co-migrate with the growing tip, suggesting that these structures are connected. We are currently probing whether sliding of the distal microtubule skeleton is responsible for pushing the MTOC vesicles forward to form and maintain the dendritic minus end distal microtubes.

He, Liu et al. (2021) International Worm Meeting "UNC- 70 (Spectrin) acts cell autonomously and non-autonomously to maintain the neuronal microtubule cytoskeleton"

Hoogenraad, Casper, He, Liu, Harterink, Martin, Meulenberg, Andrea

[International Worm Meeting, 2021]

The microtubule cytoskeleton plays a central role in neuron development and functioning. It serves as tracks for cargo transport and offers structural support to the axons and dendrites. Previously we identified a cortical anchoring complex that holds the microtubule cytoskeleton in place in neurons, where UNC-119 functions as a linker between the cortical UNC-44 (Ankyrin) and the microtubule binding UNC-33 (CRMP) (He et al., 2020). Whereas ankyrin proteins are well described scaffolds that connect various membrane proteins to the cortical spectrin-actin cytoskeleton, it is unclear how these interactions relate to the role of UNC-44 (Ankyrin) in maintaining the microtubule integrity. Here we used a floxed unc-70 (beta-Spectrin) allele for loss of function studies in a tissue specific manner. Whereas neuron specific depletion did lead to a strong reduction in its binding partner UNC-44 (Ankyrin), this did not lead to major microtubule defects. However, Spectrin depletion in both the PVD neuron and surrounding hypodermis did lead to defects in microtubule cytoskeleton immobilization and polarity organization in axons and dendrites. In agreement, we found that neuron specific depletion had only mild defect on dendritic arbors of the PVD neuron, whereas when co-depleted in the hypodermis the defects were much more pronounced. We are currently investigating whether hypodermal spectrin controls neuronal adhesion proteins such as SAX-7 (NRCAM), which in turn may bind to the UNC-44 (Ankyrin) to connect to the microtubules. Altogether, we found that UNC- 70 (Spectrin) acts cell autonomously and non-autonomously to maintain the neuronal microtubule cytoskeleton.

Harterink, Martin et al. (2013) International Worm Meeting "Neuronal microtubule organization in C. elegans."

Hoogenraad, Casper, Van den Heuvel, Sander, de Haan, Bart, Harterink, Martin

[International Worm Meeting, 2013]

In polarized cells, such as neurons, the microtubule cytoskeleton offers tracks to transport various cargos along axonal and dendritic projections. Recent data indicate that differences in microtubule organization within axons and dendrites enable molecular motors to sort cargo to specific directions and establish and maintain neuronal polarity. However how these differences are established is hardly understood. C. elegans has relatively simple neurons which offer a great starting point to understand the basics of this microtubule organization. Using GFP tagged end-binding proteins to visualize microtubule growth we have compared several classes of neurons with different characteristics (such as ciliated vs unciliated and branched vs unbrached). Here we will present a preliminary analyses of the neuronal microtubule organization of wildtype as well as in several mutants.

Harterink, Martin et al. (2017) International Worm Meeting "Microtubule nucleation from the basal body organizes the dendritic cytoskeleton for efficient distal transport."

Hoogenraad, Casper, Kapitein, Lukas, Harterink, Martin, Yau, Kah Wai, de Haan, Bart

[International Worm Meeting, 2017]

Differences in microtubule polarity organization are thought to be the evolutionary conserved determinant for axon / dendrite polarity, allowing for selective cargo transport to specific compartments. To understand how microtubules are organized in C. elegans neurons we systematically analyzed microtubule dynamics and polarity using the end binding protein EBP-2. As anticipated we found an opposite microtubule polarity between axons and dendrites allowing for selective transport into these neurites using the kinesin or dynein motors respectively (Harterink et al., 2016). Unexpectedly however, in ciliated neurons we observed a marked increase in microtubule growth events from the base of the sensory cilium (basal body) located at the dendrite tip. In addition ?-tubulin, the main microtubule nucleator, localizes to this structure suggesting that the basal body functions as a microtubule organizing center (MTOC). To address if this distal MTOC affects cargo transport we analyzed dendritic RAB-8 transport in ciliated and non-ciliated neurons. We found that only in ciliated neurons RAB-8 is efficiently transported in the distal dendrite and that inhibition of microtubule nucleation resulted in a marked decrease in distal transport. Taken together we suggest that the basal body functions as a MTOC to organize the neuronal microtubule cytoskeleton to ensure proper transport in the distal dendrite towards and from the cilium.

Harterink, Martin et al. (2015) International Worm Meeting "Distinct microtubule organizations in ciliated and non-ciliated C. elegans neurons."

Yau, Kah Wai, Harterink, Martin, Van den Heuvel, Sander, Kapitein, Lucas, de Haan, Bart, Hoogenraad, Casper

[International Worm Meeting, 2015]

In neurons, differences in microtubule organization between axons and dendrites enable selective transport of cargo into these processes. However how cargo is distributed within such processes is not well understood. This is especially important for neurons with specialized sensory endings at the dendrite tip, such as the ciliated neurons in C. elegans, which have a sensory cilium at the tip of the dendrite.We found that RAB-8, an important factor for transport to the cilium, is differently transported in ciliated dendrites. Although this is not due to differences in dendrite microtubule polarity, we found a clear difference between ciliated and non-ciliated dendrites in their microtubule organization. Ciliated dendrites have strong microtubule nucleation at the dendrite tip, whereas non-ciliated neurons do not. Furthermore two mutants which were reported to have neuronal microtubule organization defects, unc-116/KHC and unc-33/CRMP, mainly affect the dendritic microtubules of non-ciliated neurons, further supporting their organization difference. We propose a microtubule organizing center at the dendrite tip, thus having numerous microtubule tracks all the way to the cilium, allows for efficient cargo transport to and from the cilium.

He, Liu et al. (2019) International Worm Meeting "UNC-119 is part of a cortical microtubule anchoring complex, essential for neuronal polarity establishment and development."

Stucchi, Riccardo, He, Liu, Oudejans, Ellen, Harterink, Martin, Hoogenraad, Casper, Kooistra, Robbelien, Portegies, Sybren, van Leen, Eric, van Regteren Altena, Anna, de Haan, Bart

[International Worm Meeting, 2019]

The unc-119 mutant has been one of the favorite injection strains for C. elegans researchers to generate transgenic animals; the mutant severely uncoordinated which can easily be rescued. Although unc-119 is specifically expressed in neurons and its depletion leads to neuronal morphology defects, the molecular function of UNC-119 in neuron development is completely unknown. Whereas axons and dendrites are characterized by a difference in microtubule polarity enabling selective transport into each neurite, we found that depletion of unc-119 leads to loss of axon-dendrite microtubule polarity. To study the role of UNC-119 we generated a GFP knock-in line and found that the protein is diffusely localized to axons and dendrites, where it is surprisingly immobile when analyzed by FRAP. Immunoprecipitation experiments performed with the knock-in strain and HEK cells show that UNC-119 binds to both the microtubule binding protein UNC-33 (CRMP) as well as the cortical actin-spectrin organizer UNC-44 (Ankyrin), which were previously found to be important for neuronal microtubule organization (Maniar et al., 2011). This suggests a role for UNC-119 in bridging UNC-33 (CRMP) to UNC-44 (Ankyrin) to form a complex that could anchor microtubules to the cell cortex. Indeed, when analyzing microtubule dynamics using photoactivatable-GFP::TBA-1 (tubulin) the microtubules of wildtype animals are remarkably immobile, whereas in mutants for the above described genes we observed dramatic sliding of microtubules throughout the neuron. In agreement with our hypothesis for UNC-119, we were able to rescue axon-dendrite microtubule organization and the neuronal development defects of the unc-119 mutant by artificially attaching UNC-33 (CRMP) to the cell cortex. Dendritic microtubule organization has also been shown to depend on the microtubule motor UNC-116 (kinesin-1) (Yan et al., 2013), which suggests that the motor transports microtubules into the correct orientation. The depletion of unc-116 (kinesin-1) did not lead to microtubule sliding. However, depletion of unc-116 was able to suppress the microtubule sliding observed in the unc-33 mutant. This shows that in the absence of the cortical microtubule anchor complex, UNC-116 (kinesin-1) drives excessive microtubule sliding leading to microtubule organization defects. We propose that the proper development of axons and dendrites relies on a fine balance between microtubule transport and cortical microtubule stabilization.

Lockhead D et al. (2016) Mol Biol Cell "The tubulin repertoire of C. elegans sensory neurons and its context-dependent role in process outgrowth."

Barr MM, Goodman MB, Dunn AR, Lockhead D, Schwarz EM, Bellotti S, O'Hagan R, Sternberg PW, Krieg M

[Mol Biol Cell, 2016]

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division (Kapitein and Hoogenraad, 2015). They are comprised of -tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms (Gogonea et al., 1999), and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code (Fulton and Simpson, 1976). Here, we use the well-characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question, through genetic dissection of process outgrowth both in vivo and in vitro With single-cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons, and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba-1, tba-2, tbb-1, and tbb-2) function partially or fully redundantly, while two others (mec-7 and mec-12) perform specialized, context-dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies between cell types and in vivo and in vitro contexts.