Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo
- Münster University, Germany
- Weizmann Institute of Science, Israel
Figures
Figure 1 with 1 supplement
In wild type embryos the Cxcr4b receptor is uniformly distributed on the migrating PGC membrane, and its turnover is uniform around the cell circumference.
(A) A graph showing the Cxcr4b-GFP protein level measured at the front and the back (normalized to the mCherry-F') of individual migrating PGCs under conditions of endogenous Cxcl12a distribution in …
Figure 1—figure supplement 1
Functionality of the Cxcr4b tandem fluorescent timer (tft) in the context of PGC migration.
(A) Schematic representation of the cxcr4b tft RNA, showing the cxcr4b open reading frame cloned upstream to the slow maturing mCherry and to the fast maturing sfGFP, followed by nanos3′UTR that …
Figure 2 with 4 supplements
The polar positioning and number of filopodia are determined by the Cxcl12a gradient.
(A) A PGC extending filopodia during migration in wild type embryos (Video 1), and (B) in medusa (medNY054) mutant embryos lacking Cxcl12a (Video 3). (C) Filopodia distribution and number in PGCs …
Figure 2—figure supplement 1
F-actin content in filopodia extended by PGCs.
(A) A graph showing the frequency of F-actin-containing filopodia in PGCs of transgenic embryos co-expressing Lifeact-EGFP and mCherry-F'. In the cell presented on the right F-actin can be detected …
Figure 2—figure supplement 2
The procedure for cell segmentation, defining the front, back and sides of the cell.
(A) The segmentation process is performed on a 3D reconstructed image of a polarized PGC labelled with EGFP-F' and imaged by a spinning-disk microscope. (A′) Two orthogonal lines are drawn, with …
Figure 2—figure supplement 3
Filopodia do not appear to play an essential direct role in the generation of blebs.
(A) A graph showing the percentage of blebs forming relative to filopodia position (without filopodia = no filopodia on top or immediately next to bleb; with filopodia = filopodia on top or …
Figure 2—figure supplement 4
Filopodia distribution and number in PGCs knocked down for Cxcr4b.
(A) PGCs in embryos knocked down for Cxcr4b (dark bars) show apolar distribution of filopodia and an overall increased formation of these cellular protrusions as compared with control cells (light …
Figure 3 with 2 supplements
The dynamics of filopodia at the cell front are determined by the distribution of Cxcl12a.
(A) Persistence and (B) maximum length of filopodia in PGCs migrating within wild type (light bars) and medNY054 homozygous embryos (dark bars). ‘n’ indicates the number of filopodia analysed in 10 …
Figure 3—figure supplement 1
The enhanced dynamics of filopodia at the cell front is independent of bleb formation at this aspect of the cell.
(A) Persistence of filopodia at the cell front, which are not engulfed by blebs, compared to that of filopodia at the back of PGCs migrating within wild type embryos (from dataset used in Figure 3). …
Figure 3—figure supplement 2
Properties of PGCs migrating in embryos expressing a low concentration of uniform Cxcl12a.
(A) Injection of 2 pg of Cxcl12a-encoding RNA into embryos results in an increase in the proportion of the ectopic PGCs per embryo at 24 hpf. Representative embryos are shown on the right. An …
Figure 4 with 2 supplements
PGCs extend filopodia towards the chemokine source prior to cell polarization and directed migration towards the attractant.
(A, B) The cellular behaviour of PGCs (green) in response to transplanted control cells (magenta in A) or to Cxcl12a-expressing cells (magenta in B). Upper panels show the cells immediately after …
Figure 4—figure supplement 1
Polarization of PGCs encountering an artificially generated Cxcl12a gradient.
(A) Schematic experimental setup. Cells from 4 hpf medNY054 homozygous embryos expressing Cxcl12a and mCherry-F', in which Cxcr7b expression was inhibited, were transplanted into 6 hpf medNY054 …
Figure 4—figure supplement 2
PGCs extend filopodia in the direction of migration prior to polarization and actual onset of migration.
(A) Snapshots from Video 7 presenting the behaviour of a migrating cell, which makes a 90° turn (upper panel) and of a cell, which depolarizes and then migrates in the opposite direction (lower …
Figure 5 with 1 supplement
Cxcl12a internalization and interaction with filopodia.
(A) Schematic experimental setup. Cxcr7b function was knocked down in embryos, in which PGCs express mCherry on their membrane. At 16-cell stage, these embryos were injected with cxcl12a-venus RNA …
Figure 5—figure supplement 1
Cxcl12a interaction with filopodia at the front and back of the cell.
(A–A″) Cxcl12a (Venus-tagged, green) interacts with filopodia of a PGC (mCherry-F, magenta). (A′) An optical section of a PGC (a Z-projection of 4 1-µm-slices) with Cxcl12a (green) interaction with …
Figure 6 with 2 supplements
irsp53 RNA expression, Irsp53 protein localization and the role of the protein in filopodia formation.
(A) Schematic structure of the Irsp53 protein domains. The position of the mCherry fluorophore fusion at the C-terminus of the protein is presented, proteins interacting with the SH3 and CRIB …
Figure 6—figure supplement 1
Irsp53 localization within filopodia.
(A) Irsp53 can be detected in a higher proportion of filopodia at the cell front than within filopodia at the side and back of the cell. The dominant-negative version of Irsp53-mCherry cannot be …
Figure 6—figure supplement 2
Inhibition of Irsp53 does not affect bleb formation at the cell front.
(A) Number of Cxcl12a-independent blebs formed by PGCs expressing a dominant-negative version of irsp53, as compared with the number of blebs formed by control cells in medNY054 homozygous embryos. …
Figure 7 with 2 supplements
afap1l1a RNA expression, Afap1L1a protein localization and the role of the protein in filopodia formation.
(A) Schematic structure of the Afap1L1a protein domains including a serine-threonine-rich substrate domain (SD) flanked by two PH domains, a leucine zipper (Lzip), an actin-binding domain (ABD), two …
Figure 7—figure supplement 1
The effect of Afap1L1a overexpression on filopodia dynamics in PGCs migrating within a wild type environment.
(A) Persistence and (B) maximum length of filopodia in PGCs that express control RNA (light bars) or overexpress afap1l1a (dark bars). ‘n’ signifies the number of filopodia analysed in six cells …
Figure 7—figure supplement 2
Afap1L1a overexpression does not affect bleb formation at the cell front.
(A) Number of Cxcl12a-independent bleb formation by PGCs overexpressing afap1L1a, as compared with control cells in medNY054 homozygous embryos. ‘n’ signifies the number of cells analysed.
Figure 8 with 1 supplement
Filopodia are required for cellular response to polarized Cxcl12a distribution.
(A) 6–7 hpf control PGCs exhibit polarized distribution of intracellular pH as determined by the FRET efficiency of the pH sensor pH-lameleon5 protein in the cells (left). Expression of the dominant …
Figure 8—figure supplement 1
Filopodia formation is independent of the elevated pH at the cell front and of Rac1 activity in migrating PGCs.
(A) PGCs in embryos knocked down for ca15b (dark bars) show similar distribution and number of filopodia to that of control cells (light bars). (B) Examples of a control (left) and ca15b -knocked …
Figure 9 with 1 supplement
Manipulations of filopodia formation lead to PGC migration defects.
(A) Representative migration tracks of control PGCs (upper panel) and PGCs expressing the dominant-negative (dn) irsp53 version (lower panel). (B) Analysis of PGC migration tracks assessing …
Figure 9—figure supplement 1
Reducing the activity of Irsp53 has no effect on cell migration in the absence of Cxcl12a.
(A) Representative migration tracks of control PGCs (upper panel) and PGCs expressing the dominant-negative Irsp53 version (lower panel) in medNY054 homozygous embryos. (B) Analysis of PGC migration …
Figure 10
Regulation of dynamic filopodia at the cell front and their role in cell polarization and directed cell migration.
Following the activation of Cxcr4b by Cxcl12a (1), the scaffold protein Irsp53 is activated (2) and promotes the formation of dynamic filopodia at the cell front (3). The dynamic filopodia that …
Author response image 1
Author response image 2
Videos
Video 1
Migrating PGCs in wild type embryos show enhanced formation of filopodia in the direction of migration.
A 10 min time-lapse video of a PGC in kop-egfp-f'nos3′UTR embryo was captured using a 63× objective on a Zeiss AxioImager.M2 microscope equipped with a Photometrics camera (Cascade II) and VS-Laser …
Video 2
Filopodia are not directly controlling bleb formation.
Bleb-formation (asterisk) can be observed first in close proximity to filopodia (arrowheads, 10–20″) and then in a region devoid of filopodia (40″). A 40-s time-lapse video of a membrane-labelled …
Video 3
Migrating PGCs in medusa (medNY054) mutant embryos that lack Cxcl12a show enhanced apolar filopodia formation.
A 10 min time-lapse video of a PGC in medNY054 homozygous embryo was captured using a 63× objective on a Zeiss AxioImager.M2 microscope equipped with a Photometrics camera (Cascade II) and VS-Laser …
Video 4
Low concentration of uniformly expressed Cxcl12a affects cell migration, but does not immobilize the cells.
Migration tracks of PGCs in medNY054 homozygous embryos knocked down for Cxcr7b, injected with mcherry_h2b_globin3′UTR mRNA (mCherry labelling nuclei of all cells) and either with 2 pg control mRNA …
Video 5
Uniform Cxcl12a in the environment induces apolar formation of numerous long and persisting filopodia on the PGC surface.
A 10 min time-lapse video of a PGC in medNY054 homozygous embryo knocked down for Cxcr7b and that expresses uniform levels of Cxcl12a-encoding RNA was captured using a 63× objective on a Zeiss …
Video 6
PGCs extend filopodia towards the chemokine source, prior to their morphological polarization and directed migration towards cells producing the attractant.
Cells from 4 hpf medNY054 homozygous embryos, in which Cxcr7b expression was inhibited and which express mCherry-F' (red) and either control RNA (part control transplant) or express cxcl12a RNA …
Video 7
PGCs extend filopodia in the direction of migration, prior to polarization and actual onset of migration.
10 min time-lapse videos of PGCs in wild type embryos were captured using a 63× objective on a Zeiss AxioImager.M2 microscope equipped with a Photometrics camera (Cascade II) and VS-Laser Control. …
Video 8
Cxcl12a internalization.
Cxcl12a (green) is bound to the PGC membrane (magenta) and internalizes into the cell. The video was captured using a 63× objective on a Zeiss AxioImager.M2 microscope equipped with a Photometrics …
Video 9
Cxcl12a interaction with filopodia.
Cxcl12a (green) is bound to a filopodium and internalizes into the cell. An optical section of a PGC (a Z-projection of 4 1-µm-slices) is shown with Cxcl12a (green) interaction observed on the …
Video 10
Some filopodia adhere to somatic cells and appear to be under tension.
Laser-induced cut of a filopodium was performed using a Zeiss LSM710 confocal microscope equipped with a Zeiss W Plan-Apochromat 63× objective controlled by the ZEN software (Zeiss, Germany). …
Additional files
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Supplementary file 1
Experimental design, additional materials and protocols.
- https://doi.org/10.7554/eLife.05279.039
