Role of a Kinesin Motor in Cancer Cell Mechanics Kalpana Mandal, Katarzyna Pogoda, Satabdi Nandi, Samuel Mathieu, Amal Kasri, Eric Klein, François Radvanyi, Bruno Goud, Paul Janmey, Jean-Baptiste Manneville
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Kalpana Mandal, Katarzyna Pogoda, Satabdi Nandi, Samuel Mathieu, Amal Kasri, et al.. Role of a Kinesin Motor in Cancer Cell Mechanics. Nano Letters, American Chemical Society, 2019, 19, pp.7691-7702. 10.1021/acs.nanolett.9b02592. hal-02332465
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Role of a kinesin motor in cancer cell mechanics
Journal: Nano Letters
Manuscript ID nl-2019-02592e.R1
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Complete List of Authors: Mandal, Kalpana; University of Pennsylvania Perelman School of Medicine, Institute for Medicine and Engineering Pogoda, Katarzyna; Institute of Nuclear Physics Polish Academy of Sciences, Department of Experimental Physics of Complex Systems; University of Pennsylvania Perelman School of Medicine, Institute for Medicine and Engineering Nandi, Satabdi ; National Institute on Aging, Laboratory of Molecular Biology and Immunology; University of Pennsylvania, School of Veterinary Medicine Mathieu, Samuel ; Institute Curie, UMR 144 Kasri, Amal; Institute Curie, UMR 144; Pitié Salpêtrière University Hospital Neuropathology Department, ICM Brain and Spine Institute Klein, Eric ; Rutgers University Camden, Department of Biology Radvanyi, François; Institut Curie, Equipe Oncologie Moléculaire Goud, Bruno; Institut Curie, MOLECULAR MECHANISMS OF INTRACELLULAR TRANSPORT Janmey, Paul; University of Pennsylvania Institute for Medicine and Engineering, Physiology; University of Pennsylvania, Departments of Physiology and Physics & Astronomy Manneville, Jean-Baptiste; Institut Curie, MOLECULAR MECHANISMS OF INTRACELLULAR TRANSPORT
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1 Role of a kinesin motor in cancer cell mechanics 2 3 4 5 6 Kalpana Mandal1*, Katarzyna Pogoda1,2, Satabdi Nandi3,4, Samuel Mathieu5, Amal 7 8 Kasri5,6, Eric Klein7, François Radvanyi5, Bruno Goud5, Paul A Janmey1,8*, Jean- 9 10 Baptiste Manneville5* 11 12 13 1 Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia 19104, USA 14 15 2 Institute of Nuclear Physics, Polish Academy of Sciences, PL-31342 Krakow, Poland 16 3 School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA 17 4 18 Laboratory of Molecular Biology and Immunology, National Institute on Aging, Baltimore 21224, USA 19 5 Institut Curie, PSL Research University, CNRS, UMR 144, 26 rue d’Ulm, 75248 Paris cedex 05, France 20 6 21 ICM Brain and Spine Institute, Pitié Salpêtrière Hospital, 47-83 boulevard de l’Hôpital, 75013 Paris, France 22 7 Department of Biology, Rutgers University-Camden Waterfront Tech Center, NJ 08103, USA 23 8 Departments of Physiology and Physics & Astronomy, University of Pennsylvania, Philadelphia, PA 19104 24 25 26 27 * Corresponding authors: 28 29 [email protected] (K.M), [email protected], (P.A.J), jean- 30 31 [email protected] (J.-B.M) 32 33 34 35 36 37 38 Keywords: KIF20A, microrheology, optical tweezers, AFM, bladder cancer, power law, cell migration 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 ACS Paragon Plus Environment Nano Letters Page 2 of 26
Summary 1 2 Molecular motors play important roles in force generation, migration and intracellular trafficking. 3 4 Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the 5 6 kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with 7 clinical outcome. We report here a new role for the KIF20A kinesin motor protein in intracellular 8 9 mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe 10 11 cortical mechanics, we first confirm that bladder cells soften with increasing cancer grade. We then 12 13 show that inhibiting KIF20A makes the intracellular environment softer for both high and low grade 14 bladder cancer cells. Upon inhibition of KIF20A cortical stiffness also decreases in lower grade cells, 15 16 while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate 17 18 with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates 19 20 bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a 21 central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2 59 60 ACS Paragon Plus Environment Page 3 of 26 Nano Letters
Introduction 1 2 Molecular motors convert chemical energy into mechanical work to achieve active cellular processes. 3 4 The physical properties of molecular motors have been extensively studied in vitro by single 5 1–3 6 molecular approaches . In cells, assemblies of molecular motors and collective active processes 7 generate random fluctuating forces in the cytoplasm, which have recently been measured using force 8 9 spectrum microscopy4. However, the individual contribution of a given type of molecular motor to cell 10 11 mechanics is still underexplored. Kinesins form a superfamily of molecular motors that interact with 12 13 microtubules and regulate key cell functions including mitosis, cell migration and organelle transport 14 5–9. We focus here on the role of the KIF20A kinesin (also called MKLP2 for mitotic kinesin-like protein 15 16 2 or Rabkinesin-6) in the mechanics of bladder cancer cells. KIF20A is a microtubule plus-end 17 18 directed motor of the kinesin-6 family 10. KIF20A was initially shown to bind the small G protein RAB6 19 20 and to regulate retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) 21 during interphase 11. In mitosis, KIF20A localizes to the central spindle, and its phosphorylation is 22 23 required for cytokinesis 12. A recent study shows that KIF20A and myosin II interact at Golgi hotspots 24 13 25 to regulate the intracellular trafficking of RAB6-positive vesicles during interphase . KIF20A is 26 involved in the fission of transport intermediates from the Golgi apparatus and serves to anchor 27 28 RAB6 on Golgi and trans-Golgi network (TGN) membranes near microtubule nucleating sites 29 30 11,13,14. The regulation of myosin II might also be affected by KIF20A dynamics and thereby affect 31 15–17 32 force generation and intracellular microrheological properties . It was recently reported that, 33 during cortical neurogenesis, knockout of KIF20A causes the loss of neural progenitor cells and 34 35 neurons due to early cell cycle exit and neuronal differentiation 18. 36 37 KIF20A was previously reported to be highly upregulated in many cancer cell types such as 38 19–25 39 pancreatic cancer, melanoma, bladder cancer, liver cancer and breast cancer . Overexpression 40 of KIF20A is associated with increased proliferation and tumor progression, poor prognosis and 41 42 increased drug resistance in many cancers 26,27. In contrast, downregulation of KIF20A reduces cell 43 44 proliferation in pancreatic cancer and glioma cells due to cytokinesis failure, appearance of 45 binucleated cells, and apoptosis 26,28. However it has also been proposed that KIF20A could play 46 47 antagonistic roles by both activating and inhibiting tumor progression 19. Peptides derived from 48 49 KIF20A have been used to activate the immune system to kill cancer cells, which endogenously 50 29 51 express the KIF20A antigen . Hence, KIF20A has become an immunotherapeutic target for several 52 cancers, such as pancreatic or breast cancers 29–31. 53 54 Cell stiffness is often altered in pathological situations including fibrosis and cancer 32–36. In 55 36,37 56 most cancers, isolated cancer cells have been shown to be softer than their healthy counterparts . 57 58 3 59 60 ACS Paragon Plus Environment Nano Letters Page 4 of 26
Cancer cell softening is thought to be a key event during the metastatic process because more 1 2 deformable cells should migrate more efficiently through confined environments to form metastases 3 4 at a distant site from the primary tumor. Since actomyosin contractility has been shown to be crucial 5 for maintaining cell stiffness and for force generation 38,39, a large body of research has been 6 7 dedicated to study the role of actomyosin in cell mechanics 40–42. In contrast, the role of the 8 9 microtubule cytoskeleton and its associated motors in cell stiffness has been much less studied. In 10 11 particular, how microtubule motors participate to determine cell stiffness and how deregulation of 12 kinesins in disease correlates with cell mechanics are still open questions. 13 14 In this study, we investigate the mechanical role of KIF20A in bladder cancer cells using a 15 16 combination of active intracellular microrheological measurements by optical tweezers and cell 17 18 surface atomic force microscopy (AFM) indentation experiments. We focus here on the role of 19 KIF20A in regulating intracellular and cortical mechanics and correlate the mechanical effects of 20 21 KIF20A with its effects on cell migration. Our results suggest that the interaction of KIF20A with 22 23 myosin II differently regulates intracellular and cortical mechanics. The role of KIF20A in cell 24 mechanics and cell migration points to KIF20A as a potential therapeutic target for bladder cancer. 25 26 27 28 Results 29 30 31 Bladder cancer cells are softer than normal urothelial cells 32 We first compared the viscoelastic properties of primary normal human urothelial (NHU) cells and two 33 34 bladder cancer cell lines, RT112 cells and KU19-19 (KU) cells using intracellular active microrheology 35 and AFM indentation experiments (Fig. 1). RT112 cells were derived from the transitional cell 36 37 carcinoma (histological grade II) excised from a woman of unknown age, while KU cells were 38 39 established from the invasive transitional cell carcinoma of the bladder (grade III) of a 76-year-old 40 man. RT112 cells are non-invasive and tumorigenic, while KU cells are invasive and metastatic. In 41 42 addition, RT112 cells are more differentiated and do not express the epithelial–mesenchymal 43 44 transition (EMT) marker vimentin in contrast to KU cells. In the following we refer to RT112 cells as 45 43,44 46 low grade (grade II) cells and to KU cells as high grade (grade III) cells . Cells were plated on 47 crossbow shaped adhesive micropatterns to normalize their shape and intracellular organization. 48 49 Internalized 2 µm diameter beads were displaced in an oscillatory fashion using optical tweezers- 50 51 based rheology to measure the intracellular shear modulus (Fig. 1A, see Methods). We found that the 52 53 intracellular shear modulus was almost twice as high in normal NHU cells than in bladder cancer cells 54 and was significantly reduced in grade III cells compared to grade II cells (Fig. 1A). Intracellular 55 56 relaxation experiments of beads trapped by optical tweezers (Fig.1B) allowed us to measure two 57 58 4 59 60 ACS Paragon Plus Environment Page 5 of 26 Nano Letters
phenomenological parameters, the rigidity index and the bead step amplitude, and to extract the 1 2 complex shear modulus using a model based on power law rheology (see Methods). These 3 4 measurements confirmed that high grade (KU, grade III) cells are softer than low grade (RT112, 5 grade III) cells 36,37 (Fig. 1B) at the subcellular scale. AFM indentation experiments showed that 6 7 cortical stiffness is also lower in grade III cells (Fig. 1C). Note that the stiffness measured by AFM 8 9 indentation experiments was larger by two orders of magnitude than the intracellular stiffness 10 11 measured either by oscillation or relaxation experiments, suggesting that AFM measurements, but not 12 optical trapping measurements of internalized beads, are dominated by cortical stiffness. Stiffness 13 14 maps obtained on micropatterned cells show a strong spatial dispersion of stiffness values (Fig. 1D) 15 16 with higher values in the perinuclear region in both RT112 and KU cells, as observed using 17 45 18 intracellular rheology . Despite the spatial variability of the elasticity measurements, the perinuclear 19 region appears to be softer in grade III cells than in grade II cells (Fig. 1.D), consistent with the 20 21 spatially averaged data (Fig. 1C). 22 23 24 25 26 Inhibiting KIF20A softens the cell cytoplasm 27 Out-of-equilibrium active cellular processes impact strongly on cell rheology 46 . While acto-myosin 28 29 contractility is well-known to play a major role in the mechanical properties of cells and in force 30 31 generation, the role of the microtubule-associated kinesin motors in cell mechanics is much less 32 studied. Because the expression levels of several molecular motors including KIF20A have been 33 34 shown to be deregulated in bladder cancer 19, we asked whether the kinesin KIF20A could participate 35 36 in bladder cancer cell mechanics. We inhibited KIF20A by treating RT112 and KU bladder cancer 37 47 38 cells plated on glass coverslip with 50 µM paprotrain, a specific KIF20A inhibitor, for 1 hr . Cells 39 were treated with DMSO in the corresponding control experiments. Viscoelastic relaxation 40 41 experiments showed an intracellular softening of both RT112 and KU cells upon inhibition of KIF20A 42 43 (Fig. 2). The rigidity index, the bead step amplitude, and the storage modulus decrease significantly 44 45 upon treatment with paprotrain for both cell lines, while the loss modulus decreases significantly only 46 for RT112 cells (Fig. 2B-C). Interestingly, in RT112 cells, the decrease in loss modulus was larger 47 48 than that of the storage modulus (Fig. 2B). 49 50 51 52 KIF20A regulates cortical stiffness and cell motility on both soft and stiff substrates 53 To correlate intracellular rheology with the mechanical properties of the cell cortex, we measured 54 55 cortical stiffness in RT112 and KU cells by AFM indentation experiments. Surprisingly, while grade II 56 57 RT112 cells became softer when KIF20A was inhibited, grade III KU cells became stiffer (Fig. 3 A, B). 58 5 59 60 ACS Paragon Plus Environment Nano Letters Page 6 of 26
The effects of KIF20A on bladder cancer cell rheology suggest that KIF20A impacts cellular 1 2 functions that depend on cell mechanics such as cell migration. We studied individual 2D cell motility 3 4 by plating RT112 and KU bladder cancer cells on soft (G=500 Pa) polyacrylamide hydrogels or on 5 stiff (glass) substrates (Fig. 4). On both substrates, the higher-grade KU cells moved about twice as 6 7 fast as the lower grade RT122 cells (Fig. 4 A, B and supplementary movies 1 and 2). When KIF20A 8 9 was inhibited, the motility of RT112 and KU cells decreased. The speed of KU cells decreased about 10 11 3-fold and 4-fold on stiff and soft substrates respectively. In contrast, KIF20A inhibition had less effect 12 on RT112 cell motility with only a 1.5-fold and 1.1-fold decrease in cell speed on stiff and soft 13 14 substrates respectively (Fig. 4A, B and supplementary movies 3 and 4). Interestingly, both RT112 15 16 and KU bladder cancer cell lines are more motile on soft substrates than on stiff substrate, contrary to 17 48 18 other cancer cell types such as gliomas . Migration velocity as a function of substrate stiffness is 19 known to exhibit a maximal value at an optimal stiffness which depends on the number of active 20 21 molecular motors and clutches 49. In the case of bladder cancer cells, the optimal stiffness may be 22 23 shifted towards lower values. 24 25 26 27 Inhibiting KIF20A affects the subcellular localization of cortical actin and myosin II 28 29 We have shown so far that the effects of KIF20A inhibition are different in grade II RT112 cells than in 30 grade III KU cells. First, KIF20A inhibition decreases cortical stiffness in RT112 cells, whereas it 31 32 increases cortical stiffness in KU cells (Fig. 3). Second, KIF20A has stronger effects on single cell 33 34 motility in KU cells than in RT112 cells, especially on soft substrates (Fig. 4). To explain these 35 36 differences, we have first compared the expression levels and localization of KIF20A in both cell lines. 37 RT112 cells express higher levels of KIF20A than KU cells (Fig. 5A). Since RT112 and KU have 38 39 different cell cycle durations, we also measured the KIF20A expression levels in synchronized cells 40 41 and found a similar trend as in non-synchronized cells, but with higher amounts of KIF20A (Fig. 5A 42 43 and S1A). 44 Because KIF20A interacts with myosin II 13, inhibition of KIF20A may have an effect on myosin 45 46 II localization and/or cell contractility and hence cortical stiffness and cell motility. We first checked 47 48 that KIF20A inhibition does not alter the expression levels of KIF20A and myosin II (Fig. 5B and S1B) 49 and that the previously reported KIF20A/myosin II interaction also occurs in bladder cancer cells (Fig. 50 51 5C and S1C). The interaction between KIF20A and myosin II was comparable in RT112 and KU cells 52 53 but was specifically reduced in KU cells when KIF20A was inhibited (Fig. 5C and S1C). We then 54 55 asked whether KIF20A inhibition could affect KIF20A localization and the distribution of cytoskeletal 56 fibers. While KIF20A inhibition did not affect the colocalization of KIF20A with microtubules and 57 58 6 59 60 ACS Paragon Plus Environment Page 7 of 26 Nano Letters
myosin in the perinuclear region (Fig. 5D and S2), the distribution of actin and myosin II was clearly 1 2 affected in KU cells, but not in RT112 cells, with actin and myosin accumulating at the cell periphery 3 4 specifically in KU cells (Fig. 5D and S2). Such enrichment in peripheral contractile acto-myosin upon 5 KIF20A inhibition in KU cells could be responsible for the increase in cortical stiffness and the effects 6 7 on migration observed specifically in this cell line. 8 9 10 11 Discussion 12 We report here that the KIF20A kinesin motor plays a major role in bladder cancer cell mechanics. 13 14 We found that a low grade (RT112, grade II) and a high grade (KU, grade III) bladder cancer lines are 15 16 softer than normal urinary human (NHU) cells at the intracellular scale (Figure 1A), consistent with 17 36,37 18 previously published results . When KIF20A was inhibited, intracellular rigidity decreased in both 19 cell lines (Fig. 2). While a cortical softening concomitant with intracellular softening occurred in low 20 21 grade RT112 cells, high grade KU cells exhibited a stiffer cortex upon KIF20A inhibition (Fig. 3). To 22 23 our knowledge, this is the first observation of an effect of a microtubule motor on cortical mechanics. 24 25 The mechanical effects of KIF20A are mediated by its interaction with myosin II and 26 subsequent regulation of the subcellular localization of actin and myosin II. The KIF20A/myosin II 27 28 interaction was originally described to occur through Rab6 at the Golgi apparatus13. We have focused 29 30 here on the effects of KIF20A inhibition at the cell cortex, but it is likely that inhibition of KIF20A also 31 perturbs post-Golgi trafficking and more generally intracellular transport. We propose that two pools 32 33 of myosin II interact with KIF20A, a first pool localized at the Golgi apparatus and a second pool 34 35 localized at the cell cortex (Fig. S2, S3). Inhibition of KIF20A may affect the Golgi pool in both RT112 36 45 37 and KU cells and reduce intracellular stiffness by softening internal membranes such as Golgi 38 membranes where KIF20A and myosin interact. 39 40 By disrupting the interaction between KIF20A and myosin II on Golgi membranes, KIF20A 41 42 inhibition may also lead to an increase in myosin motor activity in the cytoplasm, which may be 43 44 responsible for intracellular softening. Since the elastic storage modulus 퐺’ is larger than the loss 45 modulus 퐺’’ in both bladder cancer cell lines, as generally reported in rheological measurements of 46 47 cytoplasm, the elastic behavior dominates over viscous loss. However, we observed a decrease in 48 49 loss modulus 퐺’’ upon KIF20A inhibition that is larger than the decrease in storage modulus in RT112 50 cells (Fig. 2B). The loss modulus is related to energy dissipation in the cytoplasm and thus to the 51 퐺’’ 52 effective viscosity of the cytoplasm when active non-equilibrium forces are present. We and others 53 54 have shown previously that non-equilibrium forces soften the cytoplasm by adding non Brownian 55 45,50 56 fluctuating noise to the motion of intracellular particles . Our results are thus consistent with an 57 58 7 59 60 ACS Paragon Plus Environment Nano Letters Page 8 of 26
increased activity of myosin motors in the cytoplasm upon KIF20A inhibition. Additional effects of 1 2 KIF20A inhibition on intracellular transport or microtubule dynamics, especially those of Golgi- 3 4 nucleated microtubules, may also contribute to intracellular softening. 5 In KU cells which display lower basal levels of KIF20A, the KIF20A/myosin II interaction is 6 7 decreased when KIF20A is inhibited, which may release myosin II from Golgi membranes and allow 8 9 enrichment of acto-myosin at the cortex. An increase in cortical acto-myosin density may lead to an 10 11 increase in contractility and a stiffening of the cortex and to the reduced cell motility observed 12 specifically in KU cells (Fig. 4). Because RT112 cells have a higher basal level of KIF20A and 13 14 inhibition of KIF20A does not impact its interaction with myosin II in these cells (Fig. 5 and see 15 16 discussion below), KIF20A inhibition could perturb the cortical pool of myosin II to a lesser extent and 17 18 have a lower effect on cell motility in RT112 cells. Traction force measurements could help clarify the 19 effects of KIF20A inhibition on cell-substrate adhesion and on cell motility41,51,52. 20 21 The different effects of KIF20A inhibition in grade II RT112 and grade III KU cells can likely be 22 23 explained by the difference in KIF20A expression levels (Fig. 5A) and by the different strength of the 24 previously reported KIF20A/myosin II interaction 13 (Fig. 5B-C). We found that inhibiting KIF20A did 25 26 not affect the levels of KIF20A or myosin II in either cell line (Fig. 5B) but strongly reduced the 27 28 KIF20A/myosin II interaction in KU cells but not in RT112 cells (Fig. 5C). In parallel with the reduced 29 30 KIF20A/myosin II inhibition, actin and myosin reorganized at the cortex of KU cells (Fig. 5D and Fig. 31 S3). A circular acto-myosin cortical rim appeared at the periphery of KU cells treated with the KIF20A 32 33 inhibitor replacing the lamellipodial protrusions observed in control KU cells. This finding may explain 34 35 both the loss of motility of individual KU cells and the increased cortical rigidity upon KIF20A 36 37 inhibition. Similarly, KIF20A was reported to participate in actin rearrangement and protrusion 38 formation in pancreatic cancer cells 53. In contrast no significant change in cortical acto-myosin 39 40 organization was observed in RT112 cells, which correlates with the lower effects of KIF20A inhibition 41 42 on RT112 cell motility. 43 Because microtubules play a central role in cell division, they have been early targets of 44 45 cancer drug treatments. For instance, taxol (Paclitaxel) blocks mitosis by stabilizing microtubules and 46 47 is a widely used chemotherapy drug. Similarly, microtubule-associated molecular motors are potential 48 54 49 targets for cancer therapy . Kinesins and dynein play a crucial role in many cellular functions such 50 as mitosis and cell proliferation and, not surprisingly, the expression levels of kinesins are often 51 52 modified during tumor progression. Here we have used the specific KIF20A inhibitor paprotrain to 53 54 study the effects of KIF20A inhibition on mechanical phenotypes in RT112 and KU bladder cancer 55 cells. Our results point to KIF20A as a potential target for bladder cancer therapy. KIF20A inhibition 56 57 58 8 59 60 ACS Paragon Plus Environment Page 9 of 26 Nano Letters
had stronger effects on single cell motility in KU cells than in RT112 cells (Fig. 4). The migration 1 2 velocity decreased by a factor greater than two in the case of KU cells on stiff or soft substrates (Fig. 3 4 4B). Thus, KIF20A is involved in two hallmarks of malignant transformation, hyper-proliferation and, 5 as we show here through its role in cell mechanics, increased cell motility. We can thus speculate that 6 7 a KIF20A inhibitor could be of considerable therapeutic interest as it would not only target cancer cell 8 9 proliferation but also motility and probably invasion. 10 11 12 13 Material and Methods 14 15 16 Cell culture and reagents 17 18 RT112 and KU19-19 (KU) cells were grown in RPMI medium supplemented with 10% (vol/vol) FBS 19 and 1% Penicillin/Streptomycin at 37 °C with 5% (vol/vol) CO . Primary normal human urothelium 20 2 21 (NHU) (Southgate et al., 1994) cells were grown in KSFMC medium (Life Technologies) 22 23 supplemented with 5% (vol/vol) horse serum. For intracellular rheology, cells were incubated 24 25 overnight with 2-μm-diameter fluorescently labeled latex beads (660/690 fluorescence; Bangs 26 Laboratories) before seeding them on the micropatterns. To inhibit KIF20A, cells were incubated for 27 28 at least 1 hr with 50 μM Paprotrain (Biokinesis, gift from Stéphanie Miserey-Lenkei) before 29 30 intracellular rheology and AFM measurements. 10µM Hepes was added before microrheology and 31 AFM experiments. For immunofluorescence experiments, cells were fixed with 4% (vol/vol) 32 33 paraformaldehyde. Primary antibodies were anti-β tubulin (Sigma; T4026), anti-myosin, and phalloidin 34 35 (Invitrogen), DAPI (Sigma), Myosin II (Sigma), KIF20A (Tocris bioscience). Secondary antibodies 36 37 were from Jackson Immuno Research Laboratories, E-cadherin (Cell Signaling), vinculin (Santa 38 Cruz). 39 40 41 Adhesive micropatterns 42 Crossbow-shaped micropatterns were printed on PEG-coated glass coverslips by deep UV 43 44 photolithography, and then coated with 50 μg/mL fibronectin and 20 μg/mL Alexa 546–fibrinogen- red 45 46 (Sigma). Cells were seeded on freshly prepared protein-coated micropatterns and allowed to spread 47 for at least 2–3 hrs before experiment started 55. Non-adherent cells were washed off by rinsing with 48 49 culture medium. 50 51 52 Optical tweezers-based intracellular microrheology 53 The setup combining optical tweezers and fast confocal microscopy was described in detail 54 45 55 previously . Briefly, a single fixed optical trap was built on an inverted Eclipse microscope (Nikon) 56 equipped with a resonant laser confocal A1R scanner (Nikon), a 37 °C incubator, and a nanometric 57 58 9 59 60 ACS Paragon Plus Environment Nano Letters Page 10 of 26
piezo stage (Mad City Labs). Coverslips with micropatterned or non-patterned cells containing 1 2 typically one to three internalized beads were mounted in a Ludin chamber or glass-bottom dishes 3 4 (MatTek Corporation) were used. Trapped beads were subjected either to an automated oscillatory 5 displacement of 0.5-μm amplitude (intracellular oscillations experiments) or to a 0.5 µm step 6 7 displacement (intracellular relaxation experiments). Images were recorded at 120 or 15 frames per 8 9 second in the resonant mode of the confocal scanner for 40 s using the NIS Nikon software. The 10 45 11 bead position was tracked using a homemade Matlab single particle tracking routine . 12 13 14 Intracellular relaxation experiments 15 16 We assume that the creep function 푱(풕) behaves as a power law with an exponent and a prefactor 17 휶 45 18 A, 푱(풕) = 푨풕 as described in (see methods). The bead position 푥푏(푡) was fitted using: 19 20 21 ∗ 훼 푛 ∗ 훼 푛 푡 ∞ [ ―Γ(1 + 훼)휎 퐴푡 ] 푡 ― 휏1 ∞ [ ―Γ(1 + 훼)휎 퐴(푡 ― 휏1) ] 22 푥 (푡) = 푋 ∑ ― ∑ (Eq. 1) 푏 푠휏1 푛 = 0 Γ(2 + 푛훼) 휏휏1 푛 = 0 Γ(2 + 푛훼) 23 24 25 26 We use the first four terms of Eq.1 to fit the first ten seconds of the relaxation of the bead position 푥푏 27 28 (푡) as a function of time knowing the stage step displacement 푋푠 = 0.5 µ푚 and the duration of the step 29 휏 = 40 ms (the stage step displacement is modelled by a linear increase from 0 µ푚 to 푋 = 0.5 µ푚 30 1 푠 푘 퐿 31 ∗ 푡푟푎푝 푐 32 between 푡 = 0 and 푡 = 휏1 seconds), and 휎 = 4휋푅 is a geometrical factor involving the trap stiffness 33 34 푘푡푟푎푝 = 210 pN, the bead radius 푅 = 1 µ푚 and a characteristic length 퐿푐. 35 36 The characteristic length 퐿푐 was chosen so that the generalized Stokes relation is verified 37 17,50,56,57: 퐾 = 6휋푅퐺 , where 퐺 is the shear modulus and 퐾 the apparent complex stiffness of the bead 38 39 퐾(푥푠 ― 푥푏) microenvironment. In our case, 퐺 = 휎/휖 , where 휎 = is the applied stress with 푥 and 푥 the 40 4휋푅2 푏 푠 41 42 bead and the stage displacements respectively, and 휀 = (푥푠 ― 푥푏)/퐿푐 is the strain of the 43 2 44 microenvironment. Thus, 퐺 = 퐾퐿푐/(4휋푅 ), and taking into account the generalized Stokes relation, 45 퐺 = 퐾/6휋푅, we find 퐿 = 2푅/3 = 0.67 µm and 휎 ∗ = 12.7 ± 0.5 Pa. 46 푐 47 The prefactor A and the exponent are the only two fit parameters in Eq. 1. Because bead 48 49 tracking was noisier at longer timescales due to intracellular dynamics, the data was fitted using the 50 51 following weights: 0.9, 0.09 and 0.01 for 0 휋 ′′ 훼 1 퐺 = (2휋) sin (훼2)/(퐴 Γ(1 + 훼)). (2) 2 45 3 As in , we also used a phenomenological index to quantify intracellular rigidity which we called the 4 푇 ∫0 푥푏 (푡) 푑푡 5 ‘rigidity index’ (RI) and defined as: 푅퐼 = . RI varies between 0 and 1. The bead step amplitude 6 푋푠푇 7 X corresponds to the initial displacement of the bead following the step displacement of the stage 8 b 9 (Xs=0.5 µm > Xb). 10 11 12 13 Intracellular oscillations experiments 14 The bead is initially trapped at the center of the optical tweezers at time t =0 s. An oscillating 15 16 displacement of the stage 푥푠(푡) = 푋푠 sin (휔푡) was applied at a frequency 휔 =1 Hz for 10 sec and 17 58 18 amplitude 푋푠 =0.5 μm , with 푋푏 < 푋푠 and ∆휃 the phase lag due to the viscoelastic nature of the 19 20 cytoplasm. 21 22 The force applied on the bead trapped in the optical tweezers of stiffness 푘푡푟푎푝 = 214 pN is 퐹(푡) = 퐹0 23 sin (휔푡), with 퐹 = 푘 푋 , and the corresponding bead displacement is 푥 (푡) = 푋 sin (휔푡 ― ∆휃), 24 0 푡푟푎푝 푠 푏 푏 25 where the phase lag ∆휃 is 0 for a purely elastic material and /2 for a purely viscous material. The 26 27 complex shear modulus is given by 28 퐹0 29 퐺 = 퐺′ + 푖 퐺′′ = [cos ∆휃 + 푖 sin ∆휃] 30 6휋푅푋푏 31 퐹0 Where, 퐺′ = cos ∆휃 is the storage modulus and 32 6휋푅푋푏 33 퐹0 34 퐺′′ = sin ∆휃 is the loss modulus. 35 6휋푅푋푏 36 The frequency was fixed to =1 Hz and the amplitude of the stage displacement was 푋 = 0.5 µ푚. The 37 푠 38 amplitude of the bead displacement 푋푏 and the phase lag ∆휃 were determined using sinusoidal fitting 39 40 with a homemade Matlab routine after tracking the bead position as described above. 41 42 43 AFM-based rheology 44 Atomic force microscopy (AFM)-based microindentation was performed using a Dimension Icon AFM 45 46 (ORC Bruker Nano) and a quadratic pyramid tip (Opening angle = 36°) with stiffness value 47 푘 = 0.1 푁/푚. At each indentation location, the probe tip was programmed to indent the cell at a 15 48 49 μm/s constant z-piezo displacement rate (equal rate of retraction) to an indentation depth 0.5 μm with 50 51 a maximum indentation force of 2 nN. For each cell, indentation was performed on relatively flat 52 53 regions (avoiding cell edges) of a 5 μm × 5 μm cell surface area in contact mode surface scans. 54 Force-distance indentation curves were collected from 25 different locations for each cell. For each 55 56 indentation curve, the cantilever deflection (in volts) and z-piezo displacement (in μm) were converted 57 58 11 59 60 ACS Paragon Plus Environment Nano Letters Page 12 of 26 to an indentation force (in nN) and depth (in μm) through calibrating the cantilever deflection 1 2 sensitivity (nm/V) by indenting on a hard mica substrate and a spring constant (nN/nm) via thermal 3 4 vibration. The loading portion of the curve at each location was fitted to the elastic Hertz model via 5 least-squares linear regression to calculate the effective indentation modulus at the given indentation 6 7 rate59. To determine the elasticity of the cell, every individual curve is fitted by the Hertz model. Whole 8 9 cell stiffness is determined by the width of stiffness (E) distributions when fitted with a log normal 10 ∗ 11 퐸 푡푎푛훼 2 1 distribution (Fig.3B). Force 퐹 = 2 훿 ; where E* is the effective Young’s modulus, ∗ ≈ 12 1 ― Ʋ 2 퐸 13 1 ― µ푠푎푚푝푙푒 ; = Poisson’s ratio , = face angle ; = indentation depth 14 퐸푠푎푚푝푙푒 Ʋ 훼 훿 15 16 Hydrogel preparation 17 18 For single cell random migration, cells were cultured either on glass (stiff substrate) or 500 Pa 19 polyacrylamide hydrogels (soft substrate) using previously reported methods 41,51,59. Briefly, 500 Pa 20 21 polyacrylamide hydrogels were prepared by mixing 7 % acrylamide and 0.28 % bis-acrylamide in 22 23 water. APTMS and TEMED were added to initiate gel polymerization. A sulfo-SANPAH solution in 24 25 water was added and the gel surface was activated by UV light. Gels were coated with 100 mg/ml 26 collagen type I. 27 28 29 Single cell migration 30 RT112 and KU cells were plated at a density of 30,000 cells per gel or on bare glass 22x22 mm 31 32 coverslips for 24 hrs at 37 °C with 5% (vol/vol) CO2. Cells were treated with DMSO (control) or 50 μM 33 34 paprotrain before starting time-lapse acquisition. 5 hrs to 8 hrs time lapse sequences are acquired 35 with a frame every 5mins. The Image J manual tracking plugin is used to find the cell displacement 36 37 and velocity. Velocities between two time points are obtained and averaged over the total time period. 38 39 40 Western Blot 41 KU and RT112 cells were either non treated, treated with DMSO, or treated with nocodazole (0.4 42 43 mg/ml) for 18h to synchronize the cells. Cells were lysed in a buffer containing 150 mM NaCl, 50 mM 44 45 Tris-HCl pH 7.5, and 1% Nonidet-P40 (Sigma). Protein concentrations were determined by Quick 46 Start™ Bradford 1x Dye Reagent (Bio-Rad). For Western blot analysis a nitrocellulose Protran BA 83 47 48 membrane (Life science) was used and the signal from HRP-conjugated secondary antibodies was 49 50 detected with an enhanced chemiluminescence system (ChemiDoc Touch System, Bio-Rad). 51 Quantification of the mean band intensity relative to the loading control glyceraldehyde 3-phosphate 52 53 dehydrogenase (GAPDH) was performed using the Image Lab software. 54 55 56 57 58 12 59 60 ACS Paragon Plus Environment Page 13 of 26 Nano Letters Co-Immunoprecipitation 1 2 The previously reported interaction between myosin II and KIF20A was tested by co- 3 immunoprecipitation in bladder cancer cells. RT112 and KU cells were treated with 2 mg/ml 4 5 nocodazole for 16 hrs. Cells were washed and supplemented with fresh media to recover. Cells were 6 7 either treated with DMSO or with 50 μM paprotrain for 1 hr, washed twice in PBS, and incubated on 8 9 ice for 60 min in a lysis buffer (25mM Tris pH 7.5, 50 mM NaCl, 0.1% NP40, with freshly added 10 protease and phosphatase inhibitor cocktails (Sigma). Cells were clarified by centrifuging at 10,000 x 11 12 g for 10 minutes. 500 µg of cell lysates were processed for co-immunoprecipitation using 5 µg of 13 14 KIF20A antibody coupled to dynabeads Protein A overnight at 4 °C in lysis buffer. Bead control was 15 performed using 500 ug of cell lysate without KIF20A antibody. Beads were washed four times in 1 ml 16 17 of lysis buffer followed by heating in 2x Laemmli sample buffer. The immunoprecipitates were 18 19 analyzed by SDS-PAGE followed by immunoblotting. The following primary antibodies were used: 20 21 rabbit anti-MHC (Covance; 1:2000), rabbit anti-KIF20A (Bethyl or A17425; 1:1000). Secondary 22 horseradish peroxidase (HRP)-coupled antibodies were from Jackson Laboratories. 23 24 25 Statistical analysis 26 27 Data were plotted using the Origin Pro software (OriginLab corporation) as notched box plots overlaid 28 with the data points. The box shows data between the 25th and 75th percentiles. Notches in the 29 30 notched box plots show the median and the 95% confidence interval of the median. The whiskers 31 32 correspond to outliers. When comparing two conditions (Figures 1B-C, 2, 3 and 5D) statistical 33 relevance evaluated using Student’s t-tests and p-values is indicated (n.s non significant p>0.05; * 34 35 p<0.05, ** p<0.01, *** p<0.001). In the case of multiple comparisons (Figures 1A, 4B, and 5A-C), 36 37 statistical relevance was evaluated using a one-way or a two-way ANOVA test in Origin Pro, and the 38 39 corresponding p-values are indicated. 40 41 42 43 Acknowledgements 44 We thank Stephanie Miserey-Lenkei for useful gift of Paprotrain and technical assistance of KIF20A 45 46 western blots. We thank Atef Asnacios and Charlotte Alibert for technical support and discussions. 47 48 KM was supported by a grant from MERSEC, Physical Science Oncology Center (PSOC) grant U54- 49 50 CA193417 and Labex postdoctoral fellowship. KP was funded Physical Science Oncology Center 51 (PSOC) grant U54-CA193417. SM was funded by a grant from Sorbonne Université UPMC University 52 53 Paris 06 (Programme Doctoral “Interfaces Pour le Vivant”). AK was funded by a grant from the 54 55 Agence Nationale de la Recherche (ANR). PAJ acknowledges NIH grant GM111942. This work was 56 also supported by the Center for Engineering Mechanobiology through NSF STC 1548571. JBM 57 58 13 59 60 ACS Paragon Plus Environment Nano Letters Page 14 of 26 acknowledges funding from INSERM Plan Cancer 2009-2013 INSERM - CEA Tecsan (grant number 1 2 PC201125). 3 4 5 6 References 7 8 9 (1) Rai, A. K.; Rai, A.; Ramaiya, A. J.; Jha, R.; Mallik, R. Molecular Adaptations Allow Dynein to 10 Generate Large Collective Forces inside Cells. Cell 2013, 152 (1–2), 172–182. 11 https://doi.org/10.1016/J.CELL.2012.11.044. 12 13 (2) Neuman, K. C.; Nagy, A. Single-Molecule Force Spectroscopy: Optical Tweezers, Magnetic 14 15 Tweezers and Atomic Force Microscopy. Nat. Methods 2008, 5 (6), 491–505. 16 https://doi.org/10.1038/nmeth.1218. 17 18 (3) Xiao, Q.; Hu, X.; Wei, Z.; Tam, K. Y. Cytoskeleton Molecular Motors: Structures and Their 19 Functions in Neuron. Int. J. Biol. Sci. 2016, 12 (9), 1083–1092. 20 https://doi.org/10.7150/ijbs.15633. 21 22 (4) Guo, M.; Pegoraro, A. F.; Mao, A.; Zhou, E. H.; Arany, P. R.; Han, Y.; Burnette, D. T.; Jensen, 23 M. H.; Kasza, K. E.; Moore, J. R.; et al. Cell Volume Change through Water Efflux Impacts Cell 24 Stiffness and Stem Cell Fate. https://doi.org/10.1073/pnas.1705179114. 25 26 (5) Vale, R. D. The Molecular Motor Toolbox for Intracellular Transport. Cell 2003, 112 (4), 467– 27 480. https://doi.org/10.1016/S0092-8674(03)00111-9. 28 29 (6) Vershinin, M.; Carter, B. C.; Razafsky, D. S.; King, S. J.; Gross, S. P. Multiple-Motor Based 30 Transport and Its Regulation by Tau. 2006. 31 32 (7) Exertier, P.; Javerzat, S.; Wang, B.; Franco, M.; Herbert, J. M.; Platonova, N.; Winandy, M.; 33 34 Pujol, N.; Nivelles, O.; Ormenese, S.; et al. Impaired Angiogenesis and Tumor Development by 35 Inhibition of the Mitotic Kinesin Eg5. Oncotarget 2013, 4 (12), 2302–2316. 36 https://doi.org/10.18632/oncotarget.1490. 37 38 (8) Wang, W.; Cao, L.; Wang, C.; Gigant, B.; Knossow, M. Kinesin, 30 Years Later: Recent Insights 39 from Structural Studies. Protein Sci. 2015, 24 (7), 1047–1056. https://doi.org/10.1002/pro.2697. 40 41 (9) Hirokawa, N.; Noda, Y.; Tanaka, Y.; Niwa, S. Kinesin Superfamily Motor Proteins and 42 Intracellular Transport. Nat. Rev. Mol. Cell Biol. 2009, 10 (10), 682–696. 43 https://doi.org/10.1038/nrm2774. 44 45 (10) Verhey, K. J.; Hammond, J. W. Traffic Control: Regulation of Kinesin Motors. Nat. Rev. Mol. 46 Cell Biol. 2009, 10 (11), 765–777. https://doi.org/10.1038/nrm2782. 47 48 (11) Echard, A.; Jollivet, F.; Martinez, O.; Lacapère, J. J.; Rousselet, A.; Janoueix-Lerosey, I.; Goud, 49 50 B. Interaction of a Golgi-Associated Kinesin-like Protein with Rab6. Science 1998, 279 (5350), 51 580–585. 52 53 (12) Neef, R.; Preisinger, C.; Sutcliffe, J.; Kopajtich, R.; Nigg, E. A.; Mayer, T. U.; Barr, F. A. 54 Phosphorylation of Mitotic Kinesin-like Protein 2 by Polo-like Kinase 1 Is Required for 55 Cytokinesis. J. Cell Biol. 2003, 162 (5), 863–876. https://doi.org/10.1083/jcb.200306009. 56 57 58 14 59 60 ACS Paragon Plus Environment Page 15 of 26 Nano Letters (13) Miserey-Lenkei, S.; Bousquet, H.; Pylypenko, O.; Bardin, S.; Dimitrov, A.; Bressanelli, G.; 1 Bonifay, R.; Fraisier, V.; Guillou, C.; Bougeret, C.; et al. Coupling Fission and Exit of RAB6 2 3 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions. Nat. Commun. 2017, 8 (1), 4 1254. https://doi.org/10.1038/s41467-017-01266-0. 5 6 (14) Miserey-Lenkei, S.; Chalancon, G.; Bardin, S.; Formstecher, E.; Goud, B.; Echard, A. Rab and 7 Actomyosin-Dependent Fission of Transport Vesicles at the Golgi Complex. Nat. Cell Biol. 8 2010, 12 (7), 645–654. https://doi.org/10.1038/ncb2067. 9 10 (15) Chan, C. J.; Ekpenyong, A. E.; Golfier, S.; Li, W.; Chalut, K. J.; Otto, O.; Elgeti, J.; Guck, J.; 11 Lautenschläger, F. Myosin II Activity Softens Cells in Suspension. Biophys. J. 2015, 108 (8), 12 1856–1869. https://doi.org/10.1016/j.bpj.2015.03.009. 13 14 (16) Koenderink, G. H.; Dogic, Z.; Nakamura, F.; Bendix, P. M.; MacKintosh, F. C.; Hartwig, J. H.; 15 Stossel, T. P.; Weitz, D. A. An Active Biopolymer Network Controlled by Molecular Motors. 16 17 Proc. Natl. Acad. Sci. 2009, 106 (36), 15192–15197. https://doi.org/10.1073/pnas.0903974106. 18 19 (17) Mizuno, D.; Tardin, C.; Schmidt, C. F.; Mackintosh, F. C. Nonequilibrium Mechanics of Active 20 Cytoskeletal Networks. Science 2007, 315 (5810), 370–373. 21 https://doi.org/10.1126/science.1134404. 22 23 (18) Geng, A.; Qiu, R.; Murai, K.; Liu, J.; Wu, X.; Zhang, H.; Farhoodi, H.; Duong, N.; Jiang, M.; Yee, 24 J.; et al. KIF20A/MKLP2 Regulates the Division Modes of Neural Progenitor Cells during 25 Cortical Development. Nat. Commun. 2018, 9 (1), 2707. https://doi.org/10.1038/s41467-018- 26 05152-1. 27 28 (19) Ho, J. R.; Chapeaublanc, E.; Kirkwood, L.; Nicolle, R.; Benhamou, S.; Lebret, T.; Allory, Y.; 29 Southgate, J.; Radvanyi, F.; Goud, B. Deregulation of Rab and Rab Effector Genes in Bladder 30 31 Cancer. PLoS One 2012, 7 (6), e39469. https://doi.org/10.1371/journal.pone.0039469. 32 33 (20) Zaman, M. H.; Trapani, L. M.; Sieminski, A. L.; Siemeski, A.; Mackellar, D.; Gong, H.; Kamm, R. 34 D.; Wells, A.; Lauffenburger, D. A.; Matsudaira, P. Migration of Tumor Cells in 3D Matrices Is 35 Governed by Matrix Stiffness along with Cell-Matrix Adhesion and Proteolysis. Proc. Natl. Acad. 36 Sci. U. S. A. 2006, 103 (29), 10889–10894. https://doi.org/10.1073/pnas.0604460103. 37 38 (21) Yu, Y.; Feng, Y.-M. The Role of Kinesin Family Proteins in Tumorigenesis and Progression 39 Potential Biomarkers and Molecular Targets for Cancer Therapy. Cancer 2010, 116, 5150– 40 5160. https://doi.org/10.1002/cncr.25461. 41 42 (22) Liu, S.-L.; Lin, H.-X.; Qiu, F.; Zhang, W.-J.; Niu, C.-H.; Wen, W.; Sun, X.-Q.; Ye, L.-P.; Wu, X.- 43 Q.; Lin, C.-Y.; et al. Overexpression of Kinesin Family Member 20A Correlates with Disease 44 45 Progression and Poor Prognosis in Human Nasopharyngeal Cancer: A Retrospective Analysis 46 of 105 Patients. PLoS One 2017, 12 (1), e0169280. 47 https://doi.org/10.1371/journal.pone.0169280. 48 49 (23) Imai, K.; Hirata, S.; Irie, A.; Senju, S.; Ikuta, Y.; Yokomine, K.; Harao, M.; Inoue, M.; Tomita, Y.; 50 Tsunoda, T.; et al. Identification of HLA-A2-Restricted CTL Epitopes of a Novel Tumour- 51 Associated Antigen, KIF20A, Overexpressed in Pancreatic Cancer. Br. J. Cancer 2011, 104 (2), 52 300–307. https://doi.org/10.1038/sj.bjc.6606052. 53 54 (24) Gasnereau, I.; Boissan, M.; Margall-Ducos, G.; Couchy, G.; Wendum, D.; Bourgain- 55 Guglielmetti, F.; Desdouets, C.; Lacombe, M.-L.; Zucman-Rossi, J.; Sobczak-Thépot, J. KIF20A 56 57 MRNA and Its Product MKlp2 Are Increased During Hepatocyte Proliferation and 58 15 59 60 ACS Paragon Plus Environment Nano Letters Page 16 of 26 Hepatocarcinogenesis. Am. J. Pathol. 2012, 180 (1), 131–140. 1 https://doi.org/10.1016/j.ajpath.2011.09.040. 2 3 (25) Lu, Y.; Liu, P.; Wen, W.; Grubbs, C. J.; Townsend, R. R.; Malone, J. P.; Lubet, R. A.; You, M. 4 5 Cross-Species Comparison of Orthologous Gene Expression in Human Bladder Cancer and 6 Carcinogen-Induced Rodent Models. Am. J. Transl. Res. 2010, 3 (1), 8–27. 7 8 (26) Duan, J.; Huang, W.; Shi, H. Positive Expression of KIF20A Indicates Poor Prognosis of Glioma 9 Patients. Onco. Targets. Ther. 2016, 9, 6741–6749. https://doi.org/10.2147/OTT.S115974. 10 11 (27) Lan, T.; Pang, J.; Wu, Y.; Zhu, M.; Yao, X.; Wu, M.; Qian, H.; Zhang, Z.; Gao, J.; Chen, Y. 12 Cross-Linked Hyaluronic Acid Gel Inhibits Metastasis and Growth of Gastric and Hepatic 13 Cancer Cells: In Vitro and in Vivo Studies. Oncotarget 2016, 7 (40), 65418–65428. 14 https://doi.org/10.18632/oncotarget.11739. 15 16 (28) Saito, K.; Ohta, S.; Kawakami, Y.; Yoshida, K.; Toda, M. Functional Analysis of KIF20A, a 17 Potential Immunotherapeutic Target for Glioma. J. Neurooncol. 2017, 132 (1), 63–74. 18 19 https://doi.org/10.1007/s11060-016-2360-1. 20 21 (29) Tomita, Y.; Yuno, A.; Tsukamoto, H.; Senju, S.; Kuroda, Y.; Hirayama, M.; Irie, A.; Kawahara, 22 K.; Yatsuda, J.; Hamada, A.; et al. Identification of Promiscuous KIF20A Long Peptides Bearing 23 Both CD4+ and CD8+ T-Cell Epitopes: KIF20A-Specific CD4+ T-Cell Immunity in Patients with 24 Malignant Tumor. Clin. Cancer Res. 2013, 19 (16), 4508–4520. https://doi.org/10.1158/1078- 25 0432.CCR-13-0197. 26 27 (30) Suzuki, N.; Hazama, S.; Ueno, T.; Matsui, H.; Shindo, Y.; Iida, M.; Yoshimura, K.; Yoshino, S.; 28 Takeda, K.; Oka, M. A Phase I Clinical Trial of Vaccination With KIF20A-Derived Peptide in 29 Combination With Gemcitabine For Patients With Advanced Pancreatic Cancer. J. Immunother. 30 31 2014, 37 (1), 36–42. https://doi.org/10.1097/CJI.0000000000000012. 32 33 (31) Khongkow, P.; Gomes, A. R.; Gong, C.; Man, E. P. S.; Tsang, J. W.-H.; Zhao, F.; Monteiro, L. 34 J.; Coombes, R. C.; Medema, R. H.; Khoo, U. S.; et al. Paclitaxel Targets FOXM1 to Regulate 35 KIF20A in Mitotic Catastrophe and Breast Cancer Paclitaxel Resistance. Oncogene 2016, 35 36 (8), 990–1002. https://doi.org/10.1038/onc.2015.152. 37 38 (32) Chin, L.; Xia, Y.; Discher, D. E.; Janmey, P. A. Mechanotransduction in Cancer. Curr. Opin. 39 Chem. Eng. 2016, 11, 77–84. https://doi.org/10.1016/j.coche.2016.01.011. 40 41 (33) Reid, S. E.; Kay, E. J.; Neilson, L. J.; Henze, A.-T.; Serneels, J.; McGhee, E. J.; Dhayade, S.; 42 Nixon, C.; Mackey, J. B.; Santi, A.; et al. Tumor Matrix Stiffness Promotes Metastatic Cancer 43 Cell Interaction with the Endothelium. EMBO J. 2017, 36 (16), 2373–2389. 44 45 https://doi.org/10.15252/embj.201694912. 46 47 (34) Levental, I.; Levental, K. R.; Klein, E. A.; Assoian, R.; Miller, R. T.; Wells, R. G.; Janmey, P. A. 48 A Simple Indentation Device for Measuring Micrometer-Scale Tissue Stiffness. J. Phys. 49 Condens. Matter 2010, 22 (19), 194120. https://doi.org/10.1088/0953-8984/22/19/194120. 50 51 (35) Lee, J. Y.; Chaudhuri, O. Regulation of Breast Cancer Progression by Extracellular Matrix 52 Mechanics: Insights from 3D Culture Models. ACS Biomater. Sci. Eng. 2018, 4 (2), 302–313. 53 https://doi.org/10.1021/acsbiomaterials.7b00071. 54 55 (36) Alibert, C.; Goud, B.; Manneville, J.-B. Are Cancer Cells Really Softer than Normal Cells? Biol. 56 Cell 2017, 109 (5), 167–189. https://doi.org/10.1111/boc.201600078. 57 58 16 59 60 ACS Paragon Plus Environment Page 17 of 26 Nano Letters (37) Abidine, Y.; Constantinescu, A.; Laurent, V. M.; Sundar Rajan, V.; Michel, R.; Laplaud, V.; 1 Duperray, A.; Verdier, C. Mechanosensitivity of Cancer Cells in Contact with Soft Substrates 2 3 Using AFM. Biophys. J. 2018, 114 (5), 1165–1175. https://doi.org/10.1016/j.bpj.2018.01.005. 4 5 (38) Tojkander, S.; Gateva, G.; Lappalainen, P. Actin Stress Fibers – Assembly, Dynamics and 6 Biological Roles. J. Cell Sci. 125, 1855–1864. https://doi.org/10.1242/jcs.098087. 7 8 (39) MacKintosh, F. C.; Käs, J.; Janmey, P. A. Elasticity of Semiflexible Biopolymer Networks. Phys. 9 Rev. Lett. 1995, 75 (24), 4425–4428. https://doi.org/10.1103/PhysRevLett.75.4425. 10 11 (40) Pelham, R. J.; Wang, Y. l. Cell Locomotion and Focal Adhesions Are Regulated by Substrate 12 Flexibility. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (25), 13661–13665. 13 https://doi.org/10.1073/PNAS.94.25.13661. 14 15 (41) Mandal, K.; Wang, I.; Vitiello, E.; Orellana, L. A. C.; Balland, M. Cell Dipole Behaviour Revealed 16 by ECM Sub-Cellular Geometry. Nat. Commun. 2014, 5. https://doi.org/10.1038/ncomms6749. 17 18 (42) Murrell, M.; Oakes, P. W.; Lenz, M.; Gardel, M. L. Forcing Cells into Shape: The Mechanics of 19 Actomyosin Contractility. Nat. Rev. Mol. Cell Biol. 2015, 16 (8), 486–498. 20 21 https://doi.org/10.1038/nrm4012. 22 23 (43) Marshall, C. J.; Franks, L. M.; Carbonell, A. W. Markers of Neoplastic Transformation in 24 Epithelial Cell Lines Derived from Human Carcinomas. J. Natl. Cancer Inst. 1977, 58 (6), 1743– 25 1751. 26 27 (44) Tachibana, M.; Miyakawa, A.; Tazaki, H.; Nakamura, K.; Kubo, A.; Hata, J.; Nishi, T.; Amano, 28 Y. Autocrine Growth of Transitional Cell Carcinoma of the Bladder Induced by Granulocyte- 29 Colony Stimulating Factor. Cancer Res. 1995, 55 (15), 3438–3443. 30 31 (45) Mandal, K.; Asnacios, A.; Goud, B.; Manneville, J.-B. Mapping Intracellular Mechanics on 32 Micropatterned Substrates. Proc. Natl. Acad. Sci. 2016, 113 (46), E7159–E7168. 33 https://doi.org/10.1073/pnas.1605112113. 34 35 (46) Ahmed, W. W.; Fodor, É.; Almonacid, M.; Bussonnier, M.; Verlhac, M.-H.; Gov, N.; Visco, P.; 36 37 van Wijland, F.; Betz, T. Active Mechanics Reveal Molecular-Scale Force Kinetics in Living 38 Oocytes. Biophys. J. 2018, 114 (7), 1667–1679. https://doi.org/10.1016/J.BPJ.2018.02.009. 39 40 (47) Tcherniuk, S.; Skoufias, D. A.; Labriere, C.; Rath, O.; Gueritte, F.; Guillou, C.; Kozielski, F. 41 Relocation of Aurora B and Survivin from Centromeres to the Central Spindle Impaired by a 42 Kinesin-Specific MKLP-2 Inhibitor. Angew. Chemie Int. Ed. 2010, 49 (44), 8228–8231. 43 https://doi.org/10.1002/anie.201003254. 44 45 (48) Ulrich, T. A.; de Juan Pardo, E. M.; Kumar, S. The Mechanical Rigidity of the Extracellular 46 Matrix Regulates the Structure, Motility, and Proliferation of Glioma Cells. Cancer Res. 2009, 47 69 (10), 4167–4174. https://doi.org/10.1158/0008-5472.CAN-08-4859. 48 49 (49) Bangasser, B. L.; Shamsan, G. A.; Chan, C. E.; Opoku, K. N.; Tüzel, E.; Schlichtmann, B. W.; 50 51 Kasim, J. A.; Fuller, B. J.; McCullough, B. R.; Rosenfeld, S. S.; et al. Shifting the Optimal 52 Stiffness for Cell Migration. Nat. Commun. 2017, 8, 15313. 53 https://doi.org/10.1038/ncomms15313. 54 55 (50) Guo, M.; Ehrlicher, A. J.; Jensen, M. H.; Renz, M.; Moore, J. R.; Goldman, R. D.; Lippincott- 56 Schwartz, J.; Mackintosh, F. C.; Weitz, D. A. Probing the Stochastic, Motor-Driven Properties of 57 58 17 59 60 ACS Paragon Plus Environment Nano Letters Page 18 of 26 the Cytoplasm Using Force Spectrum Microscopy. Cell 2014, 158 (4), 822–832. 1 https://doi.org/10.1016/j.cell.2014.06.051. 2 3 (51) Mandal, K.; Raz-Ben Aroush, D.; Graber, Z. T.; Wu, B.; Park, C. Y.; Fredberg, J. J.; Guo, W.; 4 5 Baumgart, T.; Janmey, P. A. Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal 6 Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force. ACS 7 Nano 2018, acsnano.8b05286. https://doi.org/10.1021/acsnano.8b05286. 8 9 (52) Roca-Cusachs, P.; Conte, V.; Trepat, X. Quantifying Forces in Cell Biology. Nat. Cell Biol. 10 2017, 19 (7), 742–751. https://doi.org/10.1038/ncb3564. 11 12 (53) Taniuchi, K.; Furihata, M.; Saibara, T. KIF20A-Mediated RNA Granule Transport System 13 Promotes the Invasiveness of Pancreatic Cancer Cells. Neoplasia 2014, 16 (12), 1082–1093. 14 https://doi.org/10.1016/j.neo.2014.10.007. 15 16 (54) Chandrasekaran, G.; Tátrai, P.; Gergely, F. Hitting the Brakes: Targeting Microtubule Motors in 17 Cancer. Br. J. Cancer 2015, 113 (5), 693–698. https://doi.org/10.1038/bjc.2015.264. 18 19 (55) Mandal, K.; Balland, M.; Bureau, L. Thermoresponsive Micropatterned Substrates for Single 20 21 Cell Studies. PLoS One 2012, 7 (5). https://doi.org/10.1371/journal.pone.0037548. 22 23 (56) Bausch, A. R.; Moller, W.; Sackmann, E. Measurement of Local Viscoelasticity and Forces in 24 Living Cells by Magnetic Tweezers. Biophys J 1999, 76 (1 Pt 1), 573–579. 25 26 (57) Ziemann, F.; Rädler, J.; Sackmann, E. Local Measurements of Viscoelastic Moduli of Entangled 27 Actin Networks Using an Oscillating Magnetic Bead Micro-Rheometer. Biophys. J. 1994, 66 (6), 28 2210–2216. https://doi.org/10.1016/S0006-3495(94)81017-3. 29 30 (58) Guet, D.; Mandal, K.; Pinot, M.; Hoffmann, J.; Abidine, Y.; Sigaut, W.; Bardin, S.; Schauer, K.; 31 Goud, B.; Manneville, J. B. Mechanical Role of Actin Dynamics in the Rheology of the Golgi 32 Complex and in Golgi-Associated Trafficking Events. Curr. Biol. 2014, 24 (15), 1700–1711. 33 https://doi.org/10.1016/j.cub.2014.06.048. 34 35 (59) Solon, J.; Levental, I.; Sengupta, K.; Georges, P. C.; Janmey, P. A. Fibroblast Adaptation and 36 37 Stiffness Matching to Soft Elastic Substrates. Biophys. J. 2007, 93 (12), 4453–4461. 38 https://doi.org/10.1529/BIOPHYSJ.106.101386. 39 40 41 42 43 Figure legends 44 45 46 47 Figure 1: Bladder cancer cells are softer than normal urothelial cells and soften with 48 increasing grade. 49 (A) Intracellular oscillations experiments. (Left) Typical fluorescent and corresponding brightfield 50 51 images of a normal human urothelial cell plated on a fibronectin-coated crossbow shape micropattern 52 53 (cyan) with an internalized bead (red). The line shows the cell contour. Scale bars, 10 μm. (Middle) 54 55 Cartoon showing the bead initially trapped at the center of the optical tweezers at time t =0 s, while 56 the cell is moved in an oscillatory fashion. Typical oscillatory displacement of the bead and fitting of 57 58 18 59 60 ACS Paragon Plus Environment Page 19 of 26 Nano Letters the curve (see Methods). (Right) Shear modulus measured by intracellular oscillating optical tweezers 1 2 microrheology in normal urothelial (NHU) cells, grade II (RT112) and grade III (KU) bladder cancer 3 4 cells. Data are from N=47, 23 and 19 cells for NHU (control cells), RT112 (grade II) and KU (grade III) 5 cells respectively. The statistical difference between the three groups is determined by a one-way 6 7 ANOVA test (* p=0.0092). (B) Intracellular relaxation experiments. (Left) Relaxation curves showing 8 9 the bead displacement from the center of the optical trap as a function of time following a 0.5 µm step 10 11 displacement of the stage for RT112 cells (blue) and KU cells (orange). The relaxation strongly 12 depends on the bead microenvironment: a stiff (blue) or soft (orange) microenvironment is 13 14 characterized by a faster or slower relaxation respectively. The inset shows an example of curve 15 16 fitting using a power-law model (see Methods). (Right) The graphs show the rigidity index and shear 17 18 modulus of RT112 and KU cells calculated from the relaxation curves using a phenomenological 19 model (for the rigidity index) or a power-law model (for the shear modulus). Data are from N=28 and 20 21 54 cells for RT112 and KU cells respectively. p-values are determined from Student’s t-test for 22 23 unpaired samples (*** p<0.0001; n.s. p=0.07 non significant). (C) AFM indentation experiments. (Left) 24 Schematic representation of the AFM experiment. A quadratic pyramid geometry tip with an opening 25 26 angle of 36° has been used for the experiment. Typical single point force-distance curve. Glass was 27 28 used for calibration. The force increases after the contact point. Glass shows an infinite stiffness 29 30 (black curve) while a cell is compliant (red curve). (middle) Distribution of the elasticity E for RT112 31 cells and KU cells (inset reprinted from 32 33 https://rifj.ifj.edu.pl/bitstream/handle/item/44/rozpr_Pogoda.pdf?sequence=1&isAllowed=y with 34 35 permission) Distributions are fitted with a log normal distribution. (Right) Elasticity of RT112 cells and 36 37 KU cells. Data are from N=60 cells per condition and for each cell about 25 force vs. distance curves 38 were acquired. p-values are determined from Student’s t-test for unpaired samples (*** p<0.0001). (D) 39 40 Spatial maps of the elasticity E measured on micropatterned cells. (Right) The white box shows the 41 42 region from which the spatial maps were taken. Scale bar, 10 µm. (Center and left) Single cell 43 stiffness maps of RT112 (center) and KU (right) cells. Note the different scales for the elasticity E 44 45 (kPa). Scale bars, 5 µm. 46 47 48 49 Figure 2: KIF20A inhibition softens the cytoplasm of both low and high grades bladder cancer 50 51 cells. (A) Average intracellular relaxation curves of control RT112 (grade II, blue) and KU (grade III, 52 orange) cells and for cells treated with the KIF20A inhibitor paprotrain (light blue, light orange). (B-C) 53 54 Rigidity index, bead step displacement, storage modulus G’ and loss modulus G’’ in control and 55 56 KIF20A inhibited RT112 cells (B) or KU cells (C). Data are from N=28 and 27 cells for control and 57 58 19 59 60 ACS Paragon Plus Environment Nano Letters Page 20 of 26 KIF20A inhibited RT112 cells and from N=53 and 51 cells for control and KIF20A inhibited KU cells. P 1 2 values are determined from Student’s t-test for unpaired samples with respect to control cells (*** 3 4 p<0.001; ** p< 0.01; * p<0.05; and n.s. p=0.112 non significant). 5 6 7 8 Figure 3: KIF20A inhibition softens the cortex of low-grade bladder cancer cells but stiffens 9 the cortex of high grade bladder cancer cells. (A) (Left) Distribution of the elasticity E measured by 10 11 AFM indentation experiments for control grade II RT112 cells (black) and for RT112 cells treated with 12 13 the KIF20A inhibitor (grey). (Right) Elasticity of control RT112 cells (blue) and RT112 cells treated 14 15 with the KIF20A inhibitor (light blue). (B) Same as (A) for grade III KU cells. Data are from N=60 cells 16 per condition and for each cell about 25 force vs. distance curves were acquired. Distributions are 17 18 fitted with a log normal distribution. p values are determined from Student’s t test for unpaired 19 20 samples with respect to control cells (*** p<0.001). 21 22 23 24 Figure 4: KIF20A inhibition reduces individual bladder cancer cell motility. (A) Rose plots of 25 randomly migrating grade II RT112 cells (upper panels) and grade KU cells (lower panels) plated on 26 27 stiff (glass, left) and soft (500 Pa polyacrylamide hydrogel, right) substrates in control conditions or in 28 29 the presence of the KIF20A inhibitor (see supplementary movies S1-S4. (B) Cell speed measured 30 from images acquired every 5 min for 5-8 hours. On stiff substrates, N=20 and 18 control and KIF20A 31 32 inhibited RT112 cells respectively, and N=17 and 22 for control and KIF20A inhibited KU cells 33 34 respectively. On soft substrates, data are from N=25 and 22 control and KIF20A inhibited RT112 cells 35 36 respectively, and N=24 and 18 control and KIF20A inhibited KU cells respectively. The statistical 37 differences between the groups are determined by a two-way ANOVA test and p-values are indicated 38 39 (*** p<0.001; n.s. p>0.05). 40 41 42 43 Figure 5: KIF20A interacts with myosin II in bladder cancer cells and its inhibition affects 44 cortical acto-myosin organization specifically in high grade bladder cancer cells. (A) (Left) 45 46 KIF20A expression in non-treated (NT), control (DMSO) or synchronized in mitosis (Sync) grade II 47 48 RT112 and grade III KU cells (top). (Right) Quantification of the blots showing the normalized 49 expression of KIF20A relative to the loading control (GAPDH). Error bars represent standard 50 51 deviation of N=3 independent experiments (results from the individual experiments are shown in 52 53 supplementary Fig. S3A). The statistical differences between the groups are determined by a two- 54 55 way ANOVA test and p-values are indicated (*** p<0.001; * p<0.05; n.s. p>0.05 non significant). (B) 56 (Left panel) Levels of myosin II and KIF20A in RT112 and KU cells treated with DMSO (control) or 57 58 20 59 60 ACS Paragon Plus Environment Page 21 of 26 Nano Letters with 50 µM paprotrain (KIF20A inhibition). (Right panel) The graph shows quantification of the KIF20A 1 2 and myosin band intensities normalized by the loading control (GAPDH or tubulin). Error bars 3 4 represent standard deviation of N=2 independent experiments (results from the individual 5 experiments are shown in supplementary Fig. S3B). The statistical differences between the groups 6 7 are determined by a two-way ANOVA test and p-values are indicated (n.s. p>0.3 non significant). (C) 8 9 Interaction between myosin II and KIF20A shown by co-immunoprecipitation in control cells treated 10 11 with DMSO and in cells treated with the KIF20A inhibitor (paprotrain 50 µM) in both RT112 and KU 12 cells. (Left panels) Endogenous myosin II is pulled down using a KIF20A antibody. Myosin II bound to 13 14 KIF20A and KIF20A were revealed by western blot analysis using an anti-Myosin II antibody and an 15 16 anti-KIF20A antibody respectively. Note that myosin II is detected in the input due to its high 17 18 expression levels as opposed to KIF20A. (Right panel) Quantification of the myosin II/KIF20A 19 interaction with respect to the total amount of KIF20A showing a reduced interaction after KIF20A 20 21 inhibition in KU cells. Error bars represent standard deviation of N=3 independent experiments 22 23 (results from the individual experiments are shown in supplementary Fig. S3C). The statistical 24 differences between the groups are determined by a two-way ANOVA test and p-values are indicated 25 26 (n.s. p>0.3 non significant). (D) Actin and myosin II localization in bladder cancer cells. (left) 27 28 Immunofluorescence images of actin (cyan, top), myosin II (red, middle) and merged actin/myosin II 29 30 (bottom) for RT112 cells (right panels) and KU cells (left panels) treated with DMSO (control) or with 31 the KIF20A inhibitor. Scale bars, 10 μm. (right) Myosin distribution was quantified by measuring the 32 33 myosin fluorescence from an outer (lamellipodium, ‘1’) region and an inner (central, ‘2’) region (left). 34 35 Normalized intensities plotted for control and KIF20A inhibited KU cells (right). N=14 and 13 cells for 36 37 control and KIF20A inhibited cells respectively. p-values are determined from Student’s t-test for 38 unpaired samples (* p<0.05). 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 21 59 60 ACS Paragon Plus Environment Figure 1 Nano Letters Page 22 of 26 Shear modulus G (Pa) 1 A x (t) x (t) p=0.0092 2 b s 0.5 * 200 3 Data Fit 4 5 150 6 0.0 7 100 8 bead 9 50 10 11 Time -0.5 0 Bead displacement (µm) Bead 0 1 2 3 4 NHU RT112 KU 12 Time (s) Displacement stage (norm.) (gr. II) (gr. III) 13 14 B15 Rigidity Index (a.u.) Shear modulus G (Pa) -7 log x 16 10 displacement b 4 -6.4 n.s. p=0.07 3.5 0.8 0.3 -6.5 *** 17 RT112 (gr. II) 3 -6.6 60 2.5 Relaxation 0.2 -6.7 18 0.4 0.3 KU (gr. III) 2 Bead displacement 1.5 -6.8 0.1 0 2 4 6 8 10 12 -1 -0.5 0 0.5 1 1.5 Time(s) 0.6 log Time(s) 19 40 826 10 20 40 0.4 21 0.2 0.2 22 20 23 0.2 24 0.0 0.1 25 0 2 4 6 8 10 0 0 Bead displacement (µm) Bead displacement (µm) Bead 26 Time (s) RT112 KU RT112 KU 27 (gr. II) (gr. III) (gr. II) (gr. III) 28 C29 Elastic modulus E (kPa) 30 3 10 *** 31 200 32 RT112 (gr. II) 8 stiff 150 33 2 KU (gr. III) 34 6 100 35 4 36 1 37 Force (nN) contact 50 2 point 38 compliant 0 39 0 0 -4000 -2000 0 2000 4000 curves of force Number 0 2 4 6 8 10 RT112 KU 40 z displacement (nm) Elasticity E (kPa) 41 (gr. II) (gr. III) 42 D43 RT112 (gr. II) KU (gr. III) 44 E (kPa) E (kPa) 45 10 5 46 47 48 49 50 51 5 µm 5 µm 52 0 0 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment PageFigure 23 of 26 2 Nano Letters A RT112 (grade II) KU (grade III) 1 0.4 0.4 2 Control Control 3 0.3 KIF20A inhib 0.3 KIF20A inhib 4 5 0.2 0.2 6 7 0.1 0.1 8 9 0 0 10 displacement (µm) Bead 0 2 4 6 8 10 (µm) displacement Bead 0 2 4 6 8 10 11 Time (s) Time (s) 12B C 13 Bead step amplitude (µm) Rigidity Index (a.u.) Bead step amplitude (µm) Rigidity Index (a.u.) 14 0.6 0.8 0.6 0.8 15 *** ** ** ** 16 0.6 0.6 17 0.4 0.4 18 19 0.4 0.4 20 0.2 0.2 21 0.2 0.2 22 23 0 0 0 0 24 Ctl KIF20A Ctl KIF20A Ctl KIF20A Ctl KIF20A 25 inhib inhib inhib inhib 26 27 Storage modulus G’ (Pa) Loss modulus G’’ (Pa) Storage modulus G’ (Pa) Loss modulus G’’ (Pa) n.s. p=0.112 28 80 * 40 ** 80 * 40 29 30 60 60 31 32 40 20 40 20 33 34 35 20 20 36 37 0 0 0 0 Ctl KIF20A Ctl KIF20A Ctl KIF20A Ctl KIF20A 38 39 inhib inhib inhib inhib 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Figure 3 B A Number of force curves Number of force curves 100 150 200 100 150 200 50 50 0 0 4 2 0 4 2 0 KU (gradeIII) RT112 (gradeII) 10 8 6 10 8 6 ACS ParagonPlus Environment KIF20A inhib KIF20A inhib Control Control Nano Letters 10 10 0 2 4 6 8 0 2 4 6 8 t KIF20A Ctl t KIF20A Ctl *** *** inhib inhib Page 24of26 PageFigure 25 of 4 26 Nano Letters 1 2 A 3 4 5 Control Control 6 80 80 80 80 7 8 40 40 40 40 9 10 0 0 0 0 11 12 -40 -40 -40 -40 13 RT112 (grade II) 14 y displacement (µm) -80 -80 -80 -80 15 -80 -40 0 40 80 -80 -40 0 40 80 -80 -40 0 40 80 -80 -40 0 40 80 16 x displacement (µm) 17 80 80 80 80 18 19 40 40 40 40 20 21 22 0 0 0 0 23 -40 -40 -40 -40 24 III) (grade KU 25 26 y displacement (µm) -80 -80 -80 -80 -80 -40 0 40 80 -80 -40 0 40 80 -80 -40 0 40 80 -80 -40 0 40 80 27 x displacement (µm) 28 29B 30 31 *** 32 3 3 *** 33 RT112 (gr. II) RT112 (gr. II) *** 34 KU (gr. III) KU (gr. III) 35 2 n.s. p=0.553 2 36 n.s. p=0.635 37 *** *** 38 1 1 39 n.s. p=0.388 40 (µm/s) Cell speed Cell speed (µm/s) 41 0 0 42 Ctl KIF20A Ctl KIF20A Ctl KIF20A Ctl KIF20A 43 inhib inhib inhib inhib 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Nano Letters Page 26 of 26 Figure 5 1 A 1.5 2 m.w. * NT RT112 (gr. II) KU (gr. III) (kDa) n.s. DMSO 3 *** Sync 130 1.0 4 KIF20A *** *** 5 100 6 GAPDH * 7 0.5 35 * 8 n.s. n.s. NT NT 9 level (a.u) KIF20A Sync Sync 0 DMSO 10 DMSO RT112 KU 11 (gr. II) (gr. III) 12 13 B m.w. 14 Control KIF20A inhib RT112 (gr. II) KU (gr. III) (kDa) 15 3 n.s. n.s. 3 250 p=0.247 16 Myosin II n.s. p=0.171 n.s. n.s. 17 2 KIF20A 2 18 100 n.s. 19 α-tubulin 1 1 20 55 21 0 (a.u) level KIF20A 22 (a.u) Myosin level 0 inhib inhib RT112 KU RT112 KU Control Control 23 KIF20A KIF20A (gr. II) (gr. III) (gr. II) (gr. III) 24 25 C 26 RT112 (gr. II) KU (gr. III) Control KIF20A inhib 27 Myosin II 0.8 n.s 28 p=0.156 29 IgG 0.6 n.s 30 KIF20A 31 0.4 n.s 32 33 Control Control Control Control Control Control 0.2 34 Myosin level (a.u) KIF20A inhib KIF20A inhib KIF20A KIF20A inhib KIF20A inhib 35 KIF20A inhib KIF20A inhib 0.0 36 Bead ctl Co-IP Input Bead ctl Co-IP Input RT112 KU 37 (gr. II) (gr. III) 38 39 D 40 RT112 (gr. II) KU (gr. III) 41 42 KU (grade III) 43 44 45 Actin 46 47 lamellipodium actin rim 48 49 50 51 In/out intensity ratio 52 1.0 Myosin II * 53 54 myosin rim 55 56 0.5 57 58 59 Merge 60 0.0 Ctl KIF20A Control KIF20A inhib Control KIF20A inhib inhib ACS Paragon Plus Environment