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. 2012 Jun 28;1(6):608-16.
doi: 10.1016/j.celrep.2012.05.013.

A motor-driven mechanism for cell-length sensing

Ida Rishal  1 , Naaman Kam, Rotem Ben-Tov Perry, Vera Shinder, Elizabeth M C Fisher, Giampietro Schiavo, Mike Fainzilber
Affiliations

A motor-driven mechanism for cell-length sensing

Ida Rishal et al. Cell Rep. .

Abstract

Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control.

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Figures

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Graphical abstract
Figure 1
Figure 1
Motor-Based Models for Cell-Length Sensing (A) A gradient-based model wherein length-encoding signals are actively transported by dynein from axon tip to cell body, with a constant rate of signal loss en route. At an early time point (T1), axons are short and signal levels at the cell body are high. At later time points (T2 and T3) the accumulating signal loss along longer tracts will reduce signal levels in the cell body. (B) Retrograde signal levels at the cell body during axon elongation from simulations of the gradient model at high (blue), medium (red), and low (green) dynein levels, respectively. Reduced dynein levels result in shorter axon lengths (e.g., using threshold indicated by horizontal line in main graph; inset). (C) A bidirectional mechanism wherein anterograde signals are transported by a kinesin from cell body to axon tip, where they activate dynein-dependent retrograde signaling to the cell body, which then represses the anterograde signal via negative feedback. (D) The model configuration of (C) generates an oscillating retrograde signal, the frequency of which decreases with axon elongation. (E and F) If axons stop growing once the signal drops below a certain frequency threshold, the simulations predict that decreasing levels of kinesin, dynein, or both motors together will lead to longer axons, as shown in (F) for growth arrest at a normalized frequency threshold of 0.02. See also Figure S1 and Movie S1.
Figure 2
Figure 2
Partial Downregulation of Certain Microtubule Motor Heavy Chains Increases Neurite Length (A and B) siRNA screen for 34 kinesin and two dynein heavy chains (n > 100). Positive hits were validated in at least three independent experiments (n > 500), showing that partial downregulation of KIF5A, KIF5B, KIF5C, KIF1B, KIF23, or DYNC1H1 increases process length up to 50%. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Student's t test and one-way ANOVA). (C) Fluorescence images of cultured DRG neurons from adult Thy1-YFP mice treated with the indicated siRNAs. Neurons were replated 1 day after siRNA transfection, and images were acquired 48 hr after replating. Scale bar, 200 μm. (D) Combined downregulation of both KIF5B and DYNC1H1 causes a greater increase in axon length than observed upon downregulation of each motor separately (n = 80; p < 0.01, ∗∗∗p < 0.001, Student's t test and one-way ANOVA). (E) Immunostaining for endogenous DYNC1H1 in DRG neurons treated with control or anti-DynHC1 siRNAs. Scale bar, 60 μm. (F) Quantification of the immunostaining over six independent experiments reveals partial downregulation of DYNC1H1 (∗∗∗p < 0.0005). (G) Immunostaining for endogenous KIF5B in DRG neurons treated with control or anti-KIF5B siRNAs. Scale bar, 60 μm. (H) Quantification of the immunostaining over three independent experiments reveals partial downregulation of KIF5B (∗∗∗p < 0.0005). (I and J) Immunostaining for endogenous Dynein HC1 in DRG neurons treated with control or anti-KIF5B siRNAs does not show any difference in endogenous levels of Dynein HC1 after siKIF5B treatment. See also Figure S2.
Figure 3
Figure 3
Increased Process Length in Sensory Neurons from a Dynein Heavy Chain 1 Mutant Mouse (A) Immunostaining of endogenous DYNC1H1 in cultured DRG neurons from wild-type and Loa/+ heterozygous mice. Scale bar, 100 μm. (B) Reduced levels of DYNC1H1 in processes of Loa/+ neurons. (C) Loa/+ DRG neurons exhibit markedly increased process length in culture when compared to wild-type neurons, as visualized by NFH staining. Scale bar, 100 μm. (D) Quantification of process lengths revealed significant differences between wild-type and Loa/+ DRG neurons in culture. ∗∗∗p < 0.001 (n = 600, Student's t test). (E) Downregulation of KIF5B in Loa/+ DRG neurons causes a greater increase in process length in culture than observed upon KIF5B downregulation in wild-type neurons. Scale bar, 200 μm. (F) Quantification of the effects of KIF5B downregulation in Loa/+ background compared to wild-type (n = 100; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, Student's t test and one-way ANOVA). (G) Electron micrographs showing immunogold labeling for DYNC1H1 on ultrathin sciatic nerve cross-sections from +/+ (left panel) and Loa/+ (right panel) mice. Scale bar, 200 nm. (H) DYNC1H1-associated gold particles within axon cytoplasm are decreased in Loa/+ mice. (I) Whole-mount neurofilament staining in E11 limbs in wild-type (+/+, left panel) and Loa/+ (right panel) mice. Scale bar, 100 μm. (J) Quantification of total nerve branch lengths reveals significant increase in total outgrowth. ∗p < 0.05 (n = 15, Student's t test). (K) Whole-mount neurofilament staining in E12 limbs in wild-type (+/+, left panel) and Loa/+ (right panel) mice. Scale bar, 200 μm. (L) Quantification of the total length of nerve branches reveals significant increase in total outgrowth. ∗p < 0.05 (n = 14, Student's t test). See also Figure S3.
Figure 4
Figure 4
Partial Downregulation of Dynein HC1 Increases Fibroblast Cell Dimensions (A) Downregulation of Dynein HC1 by siDynC1h1 in 3T3 cell cultures. (B) Quantification of 3T3 cell area over three independent experiments reveals a significant increase upon dynein downregulation, p < 0.001. (C) Quantification of cell size over three independent FACS measurements (Figure S4) revealed significant increase in total size of 3T3 cells treated with Dync1h1 siRNA. (D) Phalloidin- and DAPI-stained cultures of E14 MEFs from wild-type and Loa/+ embryos. Scale bar, 40 μm. (E) Quantification of four independent MEF cohorts reveals a significant increase in cell area in the Loa/+ cells. ∗∗∗p < 0.001. (F and G) Longest axis (yellow line) measurements of the same MEF populations show (G) a significant increase in cell length in Loa/+ MEFs as compared to wild-type. ∗∗p < 0.01. See also Figure S4.
Figure S1
Figure S1
Modeling, Related to Figure 1 (A and B) (A) Kinesin and (B) dynein velocity distributions used for the simulations (Deinhardt et al., 2006; Seitz and Surrey, 2006). (C and D) A single positive feedback loop model generates a non-informative retrograde signal. (E and F) A similar model incorporating two positive feedback steps likewise generates a non-informative retrograde signal. (G) Dominant frequency extraction in signals obtained from the composite negative feedback loop model of Figures 1C and 1D. For further details, see Extended Experimental Procedures.
Figure S2
Figure S2
Partial Downregulation of Dynein Heavy Chain 1 Does Not Affect Cytoskeleton Integrity or Growth Cone Morphology, Related to Figure 2 (A) Immunostaining for endogenous dynein heavy chain 1 (Dync1h1) in DRG neurons treated with control or anti-Dync1h1 siRNAs. Scale bar, 60 μm. (B) Quantification of the immunostainings over three independent experiments revealed partial downregulation of dynein heavy chain 1 (∗∗∗p < 0.0005). (C) Immunostaining for tubulin β3 and phalloidin-rhodamine staining for F actin did not reveal any cytoskeletal abnormalities or aberrant growth cone morphology after siRNA treatment by this protocol. Scale bar, 5 μm.
Figure S3
Figure S3
The Loa Mutation Does Not Affect Kinetics of Retrograde or Anterograde Transport in Heterozygous sensory Neurons, Related to Figure 3 (A) Electron micrographs showing co-localization of Dync1h1 and YFP near microtubules in axons of cultured DRG neurons isolated from the Thy1-YFP+/+ mouse. Dync1h1, 10 nm gold; YFP, 6 nm gold. (B and C) (B) Kinetic analysis of YFP retrograde transport or (C) of mitochondria anterograde transport in Thy1-YFP+/+ and Thy1-YFP+/Loa adult DRG neurons did not reveal any significant difference in velocity distributions.
Figure S4
Figure S4
FACS Analyses of 3T3 Cell Size, Related to Figure 4 (A) FACS analyses of 3T3 cells treated with the indicated siRNAs. The plots show forward scatter (FSC-A), a measure of cell size, versus side scatter (SSC-A), a measure of granularity. (B) Dync1h1 siRNA treatment causes an increase in cell size as shown by the right shift in population distribution in comparison to controls.
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