Filopodial Calcium Transients

 

 

Time-lapse confocal imaging of a Fluo-4 loaded Xenopus spinal neuron growth cone.  Note that the sensitivity of the detector was increased to reveal dim filopodia at the expense of saturating the central domain signal. Many short duration Ca2+ elevations (~1 sec) are localized to individual filopodia, as well as several global Ca2+ transients.  Images were acquired at 2 frames/s and time-lapse is 3.5x.  See Gomez, et al. Science 2001.

GsMtx4 stimulates axon extension in vitro

 

Globally blocking mechanically sensitive channels with GsMtx4 stimulates axon extension in vitro. Time-lapse phase contrast imaging of a Xenopus spinal cord explant culture on fibronectin. At 7.5 min, 5 uM GsMtx4 is bath applied. Note immediate veil protrusion by all growth cones and accelerated axon outgrowth. Images were acquired at 1 frame/15s and time-lapse is 128x.  See Jacques-Fricke et al. JNsc., 2006.

GsMtx4 promotes mechanotactic turning in vitro

 

Locally blocking mechanically sensitive channels with GsMtx4 promotes mechanotactic turning in vitro. Time-lapse phase contrast imaging of a Xenopus spinal neuron growth cone in a gradient of GsMtx4.  Note the micropipette loaded with 500 ┬ÁM GsMtx4 on the right generates graded GsMtx4 at the growth cone. Locally blocking mechanosensitive channels over this 15 min time-lapse sequence (128x) leads to robust axon turning toward GsMtx4.  See Kerstein, et al. JNsc, 2013.

Growth cones form point contact adhesions on Laminin, but not on non-integrin binding substrata

 

Differential targeting of tyrosine-phosphorylated proteins on integrin-binding (Laminin) versus non-integrin binding substrata (PDL). Time-lapse confocal imaging of a Xenopus spinal neuron growth cone expressing GFP-dSH2 (phosphotyrosine (PY) reporter) on PDL and LN. Note PY clusters to tips of growing filopodia of growth cones on PDL and LN, but only to point contact adhesions of growth cone on LN. Images were acquired at 1 frame/15s and time-lapse is 128x. See Robles et al., Nature Neuroscience, 2006

TMR-Kabiramide C speckling shows differential retrograde flow in growth cones on LN vs PDL

 

Time-lapse TIRF imaging of a Xenopus spinal neuron growth cones loaded with TMR-KabC and culture on LN or PDL. Note a slower rate of retrograde flow in growth cone on LN compared to PDL, which is due to formation of integrin-dependent point contact adhesions and increased clutching on LN. Images were acquired at 2 frame2/s (time-lapse 128x).  See Nichol et al., J. Neuroscience, 2016.

Actin retrograde flow is reduced as point contact adhesions in growth cones

 

TMR-KabC and GFP-dSH2 two channel imaging shows reduced retrograde flow at point contact adhesion sites of growth cones on LN. Time-lapse  two-channel TIRF imaging of a Xenopus spinal neuron growth cone expressing GFP-dSH2 and loaded with KabC. F-actin plus ends marked by KabC often appear in protrusions surrounding point contact adhesions marked by GFP-dSH2. Note that higher frame rate imaging is used to accurately track retrograde flow near adhesions. Images were acquired at 1 frame/15s (time-lapse 128x).  See Nichol et al., J. Neuroscience, 2016.

Acute BDNF treatment leads to rapid accumulation of phospho-tyrosine at tips of growing filopodia

 

Acute BDNF treatment leads to rapid accumulation of phospho-tyrosine (PY) at filopodial tips. Time-lapse confocal imaging of a Xenopus spinal neuron growth cone expressing GFP-dSH2 (PY reporter) on PDL. Note immediate accumulation of PY at the tips of existing filopodia and new filopodia that form in response to BDNF. Increased filopodial motility and new protrusions are also evident in DIC images (left). Images were acquired at 1 frame/10s and time-lapse is 60x.  See Robles et al., J. Neuroscience, 2005.

Growth cone invadosomes visualized by 3D super-resolution imaging in collagen gel

 

A Xenopus spinal neuron growth cone visualized by 3D super-resolution microscopy shows apically and basally directed invadosomes in 3D Collagen gel. Spinal neurons extending into collagen IV gel were fixed and immunolabeled for cortactin (green) and stained for F-actin (red) with phalloidin. Z-series images were collected using a structure-illumination microscope (N-SIM, Nikon) and reconstructed in 3D using Huygens essential software. Arrows indicates apically and basally directed, invadosome-like protrusions. See Santiago-Medina et al., Development, 2015.

Growth cone apical protrusion from the central domain tipped by ADAM17 MMP

 

 

3D rotation of a super resolution SIM

image of a human forebrain neuron growth cone derived from an iPSC on LNimmunolabelled for MTs (blue), F-actin (red) and ADAM17 (green). Note that

a F-actin foci in the central domain is associated with an apical protrusion that is also ADAM17 positive. Image z-series was collected on a DeltaVision OMX-SIM microscope.

Motorneuron axons generate invadosomal protrusion prior to exiting the spinal cord

 

Three-dimensional rendering of immunofluorescently labeled neurons in the embryonic Xenopus spinal cord. A 24 hpf Xenopus embryo was fixed and  immunolabeled for synaptotagmin2 (Znp1 antibody) and optically cleared. Confocal Z-series images were collected through the  volume on one side of this embryo and reconstructed using Huygens essential software. Arrows indicate invadosome-like protrusions directed peripherally toward the myotome of nascent motoneuron axons  on the ventral fascicle before exiting the CNS. Note absence of invadosomal protrusions on the dorsal fascicle. See Santiago-Medina et al., Development, 2015.