12.9: Cell Motility - Biology

12.9: Cell Motility - Biology

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

There are a number of ways in which a cell can move from one point in space to another. In a liquid medium, that method may be some sort of swimming, utilizing ciliary or flagellar movement to propel the cell. On solid surfaces, those mechanisms clearly will not work efficiently, and the cell undergoes a crawling process. In this section, we begin with a discussion of ciliary/flagellar movement, and then consider the more complicated requirements of cellular crawling.

Cilia and flagella, which differ primarily in length rather than construction, are microtubule-based organelles that move with a back-and-forth motion. This translates to “rowing” by the relatively short cilia, but in the longer flagella, the flexibility of the structure causes the back-and-forth motion to be propagated as a wave, so the flagellar movement is more undulating or whiplike (consider what happens as you waggle a garden hose quickly from side to side compared to a short piece of the same hose). The core of either structure is called the axoneme, which is composed of 9 microtubule doublets connected to each other by ciliary dynein motor proteins, and surrounding a central core of two separate microtubules.

This is known as the “9+2” formation, although the nine doublets are not the same as the two central microtubules. The A tubule is a full 13-protofilaments, but the B tubule fused to it contains only 10 protofilaments. Each of the central microtubules is a full 13 protofilaments. The 9+2 axoneme extends the length of the cilium or flagellum from the tip until it reaches the base, and connects to the cell body through a basal body, which is composed of 9 microtubule triplets arrange in a short barrel, much like the centrioles from which they are derived.

This section refers only to eukaryotes. Some prokaryotes also have motile appendages called flagella, but they are completely different in both structure and mechanism. The flagella themselves are long helical polymers of the protein flagellin, and the base of the flagellin fibers is connected to a rotational motor protein, not a translational motor. This motor (Figure (PageIndex{18})) utilizes ion (H+ or Na+ depending on species) down an electrochemical gradient to provide the energy to rotate as many as 100000 revolutions per minute. It is thought that the rotation is driven by conformational changes in the stator ring, nestled in the cell membrane.

The ciliary dyneins provide the motor capability, but there are two other linkage proteins in the axoneme as well. There are nexins that join the A-tubule of one doublet to the B-tubule of its adjacent doublet, thus connecting the outer ring. And, there are radial spokes that extend from the A tubule of each doublet to the central pair of microtubules at the core of the axoneme. Neither of these has any motor activity. However, they are crucial to the movement of cilia and flagella because they help to transform a sliding motion into a bending motion. When ciliary dynein (very similar to cytoplasmic dyneins but has three heads instead of two) is engaged, it binds an A microtubule on one side, a B microtubule from the adjacent doublet, and moves one relative to the other. A line of these dyneins moving in concert would thus slide one doublet relative to the other, if (and it’s a big “if”) the two doublets had complete freedom of movement. However, since the doublets are interconnected by the nexin proteins, what happens as one doublet attempts to slide is that it bends the connected structure instead (Figure (PageIndex{17})). This bend accounts for the rowing motion of the cilia, which are relatively short, as well as the whipping motion of the long flagella, which propagate the bending motion down the axoneme.

Although we think of ciliary and flagellar movement as methods for the propulsion of a cell, such as the flagellar swimming of sperm towards an egg, there are also a number of important places in which the cell is stationary, and the cilia are used to move fluid past the cell. In fact, there are cells with cilia in most major organs of the body. Several ciliary dyskinesias have been reported, of which the most prominent, primary ciliary dyskinesia (PCD), which includes Kartagener syndrome (KS), is due to mutation of the DNAI1 gene, which encodes a subunit (intermediate chain 1) of axonemal (ciliary) dynein. PCD is characterized by respiratory distress due to recurrent infection, and the diagnosis of KS is made if there is also situs inversus, a condition in which the normal left-right asymmetry of the body (e.g. stomach on left, liver on right) is reversed. The first symptom is due to inactivity of the numerous cilia of epithelial cells in the lungs. Their normal function is to keep mucus in the respiratory track constantly in motion. Normally the mucus helps to keep the lungs moist to facilitate function, but if the mucus becomes stationary, it becomes a breeding ground for bacteria, as well as becoming an irritant and obstacle to proper gas exchange.

Situs inversus is an interesting malformation because it arises in embryonic development, and affects only 50% of PCD patients because the impaired ciliary function causes randomization of left-right asymmetry, not reversal. In very simple terms, during early embryonic development, left-right asymmetry is due in part to the movement of molecular signals in a leftward ow through the embryonic node. This flow is caused by the coordinated beating of cilia, so when they do not work, the flow is disrupted and randomization occurs.

Other symptoms of PCD patients also point out the work of cilia and flagella in the body. Male infertility is common due to immotile sperm. Female infertility, though less common, can also occur, due to dysfunction of the cilia of the oviduct and fallopian tube that normally move the egg along from ovary to uterus. Interestingly there is also a low association of hydrocephalus internus (overfilling of the ventricles of the brain with cerebrospinal fluid, causing their enlargement which compresses the brain tissue around them) with PCD. This is likely due to dysfunction of cilia in the ependymal cells lining the ventricles, and which help circulate the CSF, but are apparently not completely necessary. Since CSF bulk flow is thought to be driven primarily by the systole/ diastole change in blood pressure in the brain, some hypothesize that the cilia may be involved primarily in ow through some of the tighter channels in the brain.

Cell crawling (Figure (PageIndex{19})) requires the coordinated rearrangement of the leading edge microfilament network, extending (by both polymerization and sliding filaments) and then forming adhesions at the new forward-most point. This can take the form of filopodia or lamellipodia,and often both simultaneously. Filopodia are long and very thin projections with core bundles of parallel microfilaments and high concentrations of cell surface receptors. Their purpose is primarily to sense the environment. Lamellipodia often extend between two lopodia and is more of a broad ruffle than a finger. Internally the actin forms more into meshes than bundles, and the broader edge allows for more adhesions to be made to the substrate. The microfilament network then rearranges again, this time opening a space in the cytoplasm that acts as a channel for the movement of the microtubules towards the front of the cell. This puts the transport network in place to help move intracellular bulk material forward. As this occurs, the old adhesions on the tail end of the cell are released. This release can happen through two primary mechanisms: endocytosis of the receptor or deactivation of the receptor by signaling/conformational change. Of course, this oversimplification belies the complexities in coordinating and controlling all of these actions to accomplish directed movement of a cell.

One model of microfilament force generation, the Elastic Brownian Ratchet Model (Mogilner and Oster, 1996), proposes that due to Brownian motion of the cell membrane resulting from continuous minute thermal fluctuation, the actin filaments that push out towards the edges of the membrane are flexed to varying degrees. If the flex is large enough, a new actin monomer can fit in between the membrane and the tip of the filament, and when the now longer filament flexes back, it can exert a greater push on the membrane. Obviously a single filament does not generate much force, but the coordinated extension of many filaments can push the membrane forward.

Once a cell receives a signal to move, the initial cytoskeletal response is to polymerize actin, building more microfilaments to incorporate into the leading edge. Depending on the signal (attractive or repulsive), the polymerization may occur on the same or opposite side of the cell from the point of signal-receptor activation. Significantly, the polymerization of new f-actin alone can generate sufficient force to move the membrane forward, even without involvement of myosin motors! Models of force generation are being debated, but generally start with the incorporation of new g-actin into a filament at its tip; that is, at the filament-membrane interface. Even if that might technically be enough, in a live cell, myosins are involved, and help to push and arrange filaments directionally in order to set up the new leading edge. In addition, some filaments and networks must be quickly severed, and new connections made, both between filaments and between filaments and other proteins such as adhesion molecules or microtubules.

How is the polymerization and actin rearrangement controlled? The receptors that signal cell locomotion may initiate somewhat different pathways, but many share some commonalities in activating one or more members of the Ras-family of small GTPases. These signaling molecules, such as Rac, Rho, and cdc42 can be activated by receptor tyrosine kinases (see RTK-Ras activation pathways, Chap. 14). Each of these has a slightly different role in cell motility: cdc42 activation leads to filopodia formation, Rac activates a pathway that includes Arp2/3 and cofilin to lamellipodia formation, and Rho activates myosin II to control focal adhesion and stress fiber formation. A different type of receptor cascade, the G-protein signaling cascade (also Chapter 14), can lead to activation of PLC and subsequent cleavage of PIP2 and increase in cytosolic Ca2+. These changes, as noted earlier, can also activate myosin II, as well as the remodeling enzymes gelsolin, cofilin, and profilin. This breaks down existing actin structures to make the cell more fluid, while also contributing more g-actin to form the new leading edge cytoskeleton.

In vitro experiments show that as the membrane pushes forward, new adhesive contacts are made through adhesion molecules or receptors that bind the substrate (often cell culture slides or dishes are coated with collagen, filaminin, or other extracellular matrix proteins). The contacts then recruit cytoskeletal elements for greater stability to form a focal adhesion (Figure (PageIndex{20})). However, the formation of focal adhesions appears to be an artifact of cell culture, and it is unclear if the types of adhesions that form in vivo recruit the same types of cytoskeletal components.

The third step to cell locomotion is the bulk movement of the cellular contents forward. The mechanisms for this phase are unclear, but there is some evidence that using linkages between the actin cytoskeleton at the leading edge and forward parts of the microtubule cytoskeleton, the microtubules are rearranged to form an efficient transport path for bulk movement. Another aspect to this may be a “corralling” effect by the actin networks, which directionally open up space towards the leading edge. The microtubules then enter that space more easily than working through a tight actin mesh, forcing flow in the proper direction.

Much of the work on microtubule-actin interactions in cell motility has been done through research on the neuronal growth cone, which is sometimes referred to as a cell on a leash, because it acts almost independently like a crawling cell, searching for the proper pathway to lead its axon from the cell body to its proper synaptic connection (A.W. Schaefer et al, Dev. Cell 15: 146-62, 2008).

Finally, the cell must undo its old adhesions on the trailing edge. This can happen in a number of different ways. In vitro, crawling cells have been observed to rip themselves off of the substrate, leaving behind tiny bits of membrane and associated adhesion proteins in the process. The force generated is presumed to come from actin-myosin stress fibers leading from the more forward focal adhesions. However, there are less destructive mechanisms available to the cells. In some cases, the adhesivity of the cellular receptor for the extracellular substrate can be regulated internally, perhaps by phosphorylation or dephosphorylation of a receptor. Another possibility is endocytosis of the receptor, taking it off the cell surface. It could simply recycle up to the leading edge where it is needed (i.e. transcytosis), or if it is no longer needed or damaged, it may be broken down in a lysosome.

Microtubule Dynamics in Mitotic Spindle Displayed by Polarized Light Microscopy

The first sequence shows an endosperm cell from the African blood lily, Haemanthus katherinae, undergoing mitosis (Figure 1). This sequence, captured by A.S. Bajer and J. Molé-Bajer using phase-contrast microscopy, was observed in cells that had been flattened between a layer of agar and gelatin to improve their visibility (Bajer and Molè-Bajer, 1956, 1986). The sequence vividly displays the chromosomes as they condense and align on the metaphase plate (Figure1b). In the meantime the three large, dark nucleoli (Figure 1a) disappear. Then the chromosomes split and move apart in anaphase (Figure 1c). Finally the chromosomes become decondensed as they are packaged into two daughter nuclei in telophase (Figure 1d). Between the nuclei, small dancing vesicles appear (Figure 1c), align, and fuse with each other to form the cell plate (Figure 1d). The cell plate eventually gives rise to the cell walls and separates the plant cell into two.

Fig. 1. Mitosis and cell plate formation in a flattened endosperm cell of the African blood lily, Haemanthus katherinae, observed with phase contrast microscopy.

In the next sequence, we see the pollen mother cell of an Easter lily,Lilium longiflorum, undergoing mitosis and cell division (Figure 2). These cells synchronously undergo the first of their two divisions to form four pollen grains when the flower bud is exactly 22.4 mm long (Figure3). A bud of this length was collected and centrifuged at ∼1800 × g for 3 min to displace the highly light-scattering granules and to make the other contents of the cell more visible. After excising an anther from the centrifuged flower bud in seven-eighths-strength frog Ringer’s solution, the cells were observed between crossed polarizers in the presence of a compensator (Figure 4). Observed with a polarizing microscope in this manner, regions of the cell where molecules are regularly aligned, i.e., birefringent regions, become highlighted (Figures 2, 5, and 6).

Fig. 2. Mitosis and cell plate formation in centrifuged pollen mother cell of the Easter lily, Lilium longiflorum, observed with polarization microscopy. Reproduced from The Journal of Cell Biology, vol 130, 687–700, 1995, by copyright permission of The Rockefeller University Press.

Fig. 3. Length of flower bud of Lilium longiflorum in which pollen mother cells undergo their first division (after Erickson, 1948).

Fig. 4. Schematic of a polarizing microscope with crossed polarizers and a compensator. Reproduced from The Journal of Cell Biology, vol 139, 985–994, 1997, by copyright permission from The Rockefeller University Press.

As with the Bajer’s endosperm cell series, this series on the pollen mother cell mitosis was initially time-lapse recorded on 16-mm ciné film. The elapsed time from breakdown of the nuclear envelope to formation of the cell plate was ∼2 h. The cinérecords were transferred to video some 40 years later.

The polarizing microscope view of the pollen mother cells distinctly shows the spindle fibers that were not visible with phase-contrast microscopy (for polarizing microscope images of Haemanthusendosperm cells, see Inoué and Bajer, 1961 Inoué, 1964). Phase-contrast microscopy clearly shows the chromosome and nucleoli because of their higher refractive index, but not the spindle fibers that lead the chromosomes apart to the spindle poles or the phragmoplast fibers that bring the vacuoles to the cell plate. The refractive index of these fibers is too close to that of the surrounding cytoplasm. They nevertheless show clearly in a well-tuned polarizing microscope, because the fibers are birefringent, being made up of a bundle of regularly aligned molecular filaments. This sequence, taken by Inoué in 1950, demonstrated, for the very first time, the reality of spindle fibers and fibrils in living cells (Inoué, 1953, 1964) as well as the highly dynamic, labile nature of the molecular filaments (later identified as microtubules).

The microtubules disassembled reversibly when cells were exposed to cold, to high hydrostatic pressure, or to antimitotic drugs such as colchicine (reviewed in Inoué, 1964, 1981). During slow depolymerization of microtubules by these agents, metaphase-arrested chromosomes were pulled to a spindle pole anchored to the cell surface. After removal of the depolymerizing agent, growing spindle fibers pushed the chromosomes toward the metaphase plate. Thus arose the notion that chromosome movement toward the metaphase plate was associated with (and powered by) assembly and growth of microtubules, whereas movement of the chromosomes toward the spindle poles was associated with (and powered by) disassembly and shortening of the microtubules attached to the kinetochore of each sister chromosome (recent evidence and discussions summarized in Inouéand Salmon, 1995).

These polarized light microscopy studies on the birefringence of dividing cells demonstrated the assembly properties of microtubules and their dynamic function in living cells long before microtubules themselves were discovered or their assembly properties were characterized in vitro (reviewed in Inoué, 1981).

The microtubules that make up the spindle fibers and their shortening in anaphase can be seen more distinctly in the sequence of high-resolution images of a grasshopper spermatocyte (Pardalophora apiculata Figure5) taken by Nicklas (1971) with a rectified polarizing microscope. Rectification provides a higher-resolution image, restoring the needed extinction and correcting for the image error found in conventional polarizing microscopes when high–numerical aperture lenses are used (Inoué and Hyde, 1957).

Fig. 5. Primary spermatocyte of Pardalophora apiculata observed with a rectified polarizing microscope (fromNicklas, 1971). Reproduced from Advances in Cell Biology, vol 2, 225–298, copyright 1971 Appleton-Century-Crofts.

The final video sequence shows a dividing newt (Taricha granulosa) lung epithelial cell (Figure6) recorded by R. Oldenbourg, P.T. Tran, and E.D. Salmon with the new Pol-Scope. With the Pol-Scope, each image is generated by an image-processing computer from four video images taken in rapid succession at different settings of two electronically driven liquid crystal compensators. In the images thus displayed by the computer, the brightness of each pixel is strictly proportional to the birefringence of the specimen point and independent of orientation of the birefringence axis (Oldenbourg, 1996). Thus, in addition to providing displays with exquisitely high resolution and definition, Oldenbourg’s new Pol-Scope provides highly sensitive, dynamic image information on molecular alignment that is strictly quantitative.

Fig. 6. Mitosis in tissue-cultured lung cell of a newt,Taricha granulosa, recorded with the new Pol-Scope. Reproduced from The Journal of Cell Biology, vol 139, 985–994, 1997, by copyright permission of The Rockefeller University Press.

In addition to being of historic interest, considered use of polarized light microscopy should continue to reveal much regarding the behavior of molecular and fine structural dynamics, noninvasively, in dividing, developing, and otherwise actively functioning living cells (Oldenbourg, 1998).

Progesterone (P4) Receptor Membrane Component 1 (PGRMC1) is a cytochrome b5-related heme-binding protein with multiple functions including interaction with cytochrome P450 enzymes. Briefly, (the reader is referred to previous reviews) non-comprehensive PGRMC1 functions include membrane trafficking, P4 responsiveness and steroidogenesis, fertility, lipid transport, neural axon migration, synaptic function, and anti-apoptosis. Its subcellular localization can be cytoplasmic, nuclear/nucleolar, mitochondrial, endoplasmic reticulum, cytoplasmic vesicles, or extracellular [1,2,3]. It is involved in cell cycle processes at the G1 checkpoint and during mitosis [4,5,6,7,8,9], and elevated PGRMC1 expression has been associated with poor prognosis in multiple types of cancer [2, 10,11,12,13,14,15].

Predicted binding site motifs for Src homology 2 (SH2) and Src homology 3 (SH3) proteins in PGRMC1 can potentially be negatively regulated by phosphorylation at adjacent casein kinase 2 (CK2) consensus sites [2, 16, 17]. However, while CK2 knockdown leads to reduced phosphorylation of the corresponding C-terminal CK2 site of PGRMC2, PGRMC1 phosphorylation at S181 was unaffected by CK2 knockout in C2C12 mouse myoblast cells [18]. These and Y180 can all be phosphorylated in vivo, and constitute a potential regulated signaling module [19].

We hypothesized that PGRMC1 is a signal hub protein with wide ranging effects on cell biology [2, 19,20,21]. The highly conserved motif at Y180/S181 phosphorylation arose early in animal evolution concurrently with the embryological organizer of gastrulation (e.g. Spemann-Mangold organizer), and prior to the evolution of deuterostomes [20, 22]. Since all of the descendants of the organism that first acquired S181 exhibit bilateral body symmetry, this mutation predated the rules governing cell growth and division leading to bilateral body plan and more types of tissues. The mechanisms that generate and maintain cell- and tissue-type differentiation states are very often perturbed in cancer [23]. Therefore, PGRMC1 tyrosine phosphorylation could potentially strongly influence mammalian cell function and differentiation status.

This present study was prompted by our discovery of differential PGRMC1 phosphorylation status between estrogen receptor-positive and -negative breast cancers [24]. PGRMC1 was induced in the hypoxic zone of ductal carcinoma in situ breast lesions at precisely the time and place that cells require a switch to glycolytic metabolism known as the Warburg effect, leading us to predict a Warburg-mediating role for PGRMC1. Furthermore, a PGRMC1 S57A/S181A double CK2 site mutant (DM, Fig. 1a) enabled the survival of peroxide treatment [24]. Sabbir [25] recently reported that PGRMC1 induced a P4-dependent metabolic change resembling the Warburg effect in HEK293 cells, which was associated with changes in PGRMC1 stability, post-translational modifications, and subcellular locations. PGRMC1 regulation of glucose metabolism is supported by its implicated mediation of the placental P4-dependent shift from aerobic towards anaerobic glucose metabolism in gestational diabetes [26], association with the insulin receptor and glucose transporters [27], and probable involvement (based upon AG-205 sensitivity) in P4-mediated increase in neuronal glycolysis [28].

MIA PaCa-2 pancreatic cancer cells morphology is affected by PGRMC1 phosphorylation status. a PGRMC1-HA proteins constructed for this figure. TMH: Trans-membrane helix. HA: the C-terminal 3x hemaglutinin tag. b Detection of exogenous PGRMC1 expression levels by western blot (upper panel). Equal loading is controlled by quantifying beta actin (lower panel). The results show three totally independent stably transfected cell lines per plasmid from (A). Open arrow: Exogenous PGRMC1-HA (Ex.). Shaded arrow: endogenous PGRMC1 (End.). Filled arrow: beta actin. The molecular weight ladder is Bio-Rad 1610377 Dual Xtra Standards. c Box plots quantification of replicate gels of (B) with signals normalized to beta actin from the same respective lanes. n = 4 lanes for MP and n = 6 for WT, DM and TM (replicates of respective lines 1–3 per condition). There were no significant differences (ns) except for the exogenous band in MP (ANOVA, post-hoc Dunnet’s T3). d Western blot quantification of HA-tagged exogenous PGRMC1, following B but detecting PGRMC1 with anti-HA antibody. The molecular weight ladder is Abcam ab116028 Prestained Protein Ladder. e PGRMC1 mutant protein expression alters MIA PaCa-2 cell morphology. PGRMC1-HA-expressing stable cells (respective lines 1 from B) or MP cells were stained with a FITC-tagged anti-HA antibody (Anti-HA) and imaged by confocal microscopy. DNA was stained with DAPI. Cells were also imaged in differential interference contrast (DIC) microscopy mode. The respective left panels show merged images of all 3 channels. f The rounded phenotype of double and triple mutant (E) was reversed to elongated phenotype after 125 μM ROCKI addition, but not by addition of DMSO vehicle control

We previously observed that MIA PaCa-2 pancreatic cancer (MP) cells [29,30,31,32] exhibited marked morphological and metabolic changes when the DM protein was expressed [33]. MP cells exist in culture as a mixed adherent population of elongated “fibroblast-shaped” morphology, a minority population of rounded morphology with bleb-like protrusions, and some multicellular clumps, as well as some rounded suspension cells. They have undergone epithelial-mesenchymal transition [34], and can further undergo mesenchymal-amoeboid transition (MAT), which requires Rho Kinase- (ROCK)- dependent morphological change from “elongated” mesenchymal cells to rounded amoeboid cells [35].

Here, we mutagenically examined the effects of altered PGRMC1 phosphorylation status on MP cells to gain insights into PGRMC1-dependent signaling in vitro, revealing an important role for Y180. Our objectives were to initially characterize the effects of mutations at the two DM putative negative regulatory phosphorylation sites, S57 and S181, on cell biology, as well as mutating Y180 which forms the center of a putative SH2 target motif to test the hypothesis that the DM effect required Y180. Importantly this investigation was extended to an in vivo system, specifically a subcutaneous mouse xenograft tumorigenesis model, in order to address the requirement of Y180 for tumor development in a complex environment. In a companion publication [36], we describe differences in metabolism, genomic mutation rates, and epigenetic genomic CpG methylation levels associated with the PGRMC1 phosphorylation mutants described in this present study.


Generation of Cell Lines

Rat1 fibroblasts were transfected with the use of LipofectAMINE PLUS (Life Technologies, Rockville, MD) with 1.05 μg of pPUR (Clontech, Palo Alto, CA) only or cotransfected with 0.05 μg of pPUR and 1.0 μg of pKH3-p190RhoGAP or pKH3-p190RhoGAP R1283A (Tatsis et al., 1998) and then selected in 10 μg/ml puromycin (Sigma, St. Louis, MO). Clonal lines were established and screened for expression by immunoblotting with monoclonal antibodies against hemagglutinin epitope (HA Covance, Richmond, CA) and p190RhoGAP (BD Transduction Laboratories, Lexington, KY). Ten pPUR clones (mock), five HA-p190RhoGAP R1283A clones (p190-RA), and three HA-p190RhoGAP clones (wt-p190) of similar expression levels were pooled. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies), antibiotics, and 10 μg/ml puromycin.

For all experiments, cells were replated in the absence of puromycin the day before experiments, trypsinized, washed twice in DMEM, and then suspended before plating for 1 h in DMEM and 0.5% fatty acid-free bovine serum albumin (Sigma). Plating surfaces were coated with 20 μg/ml fibronectin (Life Technologies) overnight at 4°C, followed by blocking for 1 h with 0.5% fatty acid-free bovine serum albumin. RhoA inhibition was achieved by introduction of C3 transferase as previously described (Renshaw et al., 1996). Briefly, 12 μg (spreading assays) or 34 μg (RhoA assays) of C3 transferase or glutathione S-transferase (GST as a negative control) were mixed with 25 μl of LipofectAMINE and 200 μl of DMEM for 15 min and then added to a 10-cm dish of subconfluent cells in 5 ml of DMEM for 90 min. RhoA was activated by transfecting cells with 0.5 μg of PAX142-LscD4-HA (Whitehead et al., 1996), an expression vector encoding a constitutively active mutant of the RhoA exchange factor, Lsc. As a negative control for transfection, cells were transfected with pEGFP-C1 (Clontech).

RhoA Activity Assay

RhoA activity was measured with the use of a technique similar to the method described by Ren et al. (1999). Briefly, cells were lysed in 300 μl of 50 mM Tris, pH 7.4, 10 mM MgCl2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 10 μg/ml each of aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 200 μM vanadate. Lysates (500–750 μg) were cleared at 16,000 × g for 5 min, and the supernatant was rotated for 30 min with 30 μg of GST-RBD (GST fusion protein containing the RhoA-binding domain [amino acids 7–89] of Rhotekin) bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Samples were washed three times in 50 mM Tris, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% Triton X-100, and inhibitors and then immunoblotted with RhoA monoclonal antibodies (BD Transduction Laboratories). Whole cell lysates were also immunoblotted for RhoA as loading controls.


Cells on coverslips were fixed for 15 min in 3.7% formaldehyde and then permeabilized for 5 min in 0.5% Triton X-100. Texas Red-phalloidin and Hoechst 33342 from Molecular Probes (Eugene, OR) were used to label F-actin and nuclei, respectively. Monoclonal antibodies against the HA (Covance) or GM130 (BD Transduction Laboratories) were used, followed by incubation with fluorescein isothiocyanate-donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were obtained on an Axiophot microscope (Zeiss, Thornwood, NY) with the use of a MicroMAX 5-MHz cooled charge-coupled device camera (Princeton Instrument, Trenton, NJ) and Metamorph Image software (Universal Imaging, West Chester, PA).

Spreading Assay

Suspended cells were plated on fibronectin-coated coverslips for 10, 20, 30, 45, 60, and 180 min. Coverslips were fixed and stained with Coomassie blue (2% Brilliant Blue [Sigma], 45% methanol, and 10% acetic acid) for 10 min and then washed with water and mounted. The relative areas of individual cells from Metamorph images were quantified with the use of National Institutes of Health Image software. Data from these and all other experiments were considered significantly different if the p values, as determined by two-tailedt-tests, were <0.05.

Migration Assay

The top and bottom surfaces of 8-μm pore, 6.5-mm polycarbonate Transwell filters (Corning Costar, Corning, NY) were coated with fibronectin. Cells (2.0 × 10 4 ) were seeded on the upper surface of the filters and migration was allowed to proceed for 3 h. Cells were fixed, stained with Coomassie blue, and the number of cells per 25× field on the lower surface of the filters was counted. For some experiments Rho kinase was inhibited with 3 μM Y-27632 (Uehata et al., 1997b Welfide, Osaka, Japan).

Monolayer Wound Healing and Polarity Assay

Cells (4.0 × 10 5 ) in serum-free medium were seeded on fibronectin-coated coverslips in 24-well plates and allowed to adhere for 1 h. The monolayer was then wounded with a rubber policeman. Cells were washed once, fresh serum-free media was added, and the cells were allowed to invade the wound for 2 h (polarity assay) or 3 h (morphology assay) before fixation. Nuclei, F-actin, and the Golgi were labeled as described above with Hoechst 33342, phalloidin, and GM130 antibodies, respectively. Cells bordering the wound were considered polarized if the Golgi was orientated toward the wound-side of the nucleus (Kupfer et al., 1982).

Novel insights into how autophagy regulates tumor cell motility

Metastasis requires tumor cells to overcome a series of challenges to successfully travel to and colonize new microenvironments. As an adaptive (or maladaptive) response to stress, macroautophagy/autophagy has garnered increasing interest with respect to cancer metastasis, supported by clinical observations of increased autophagic flux in distant metastases relative to primary tumors. Recently, we identified a new role for autophagy in tumor cell motility through the turnover of focal adhesions, large multi-protein structures that link extracellular matrix-bound integrins to the cytoskeleton. The disassembly of focal adhesions at the cell rear is critical to forward movement and successful migration/invasion. We demonstrated that the focal adhesion protein PXN (paxillin), which serves as a crucial scaffolding and signal integrator, binds directly to LC3B through a conserved LC3-interacting region (LIR) motif to stimulate focal adhesion disassembly and metastasis and that this interaction is further promoted by oncogenic SRC.

We identified a specific requirement for autophagy in cell motility during metastasis that is independent of effects on proliferation or viability using an orthotopic murine model of metastatic mammary cancer. Unlike in many other models, the inhibition of autophagy has no effect on primary tumor growth, possibly due to the highly aggressive nature of the tumor model employed. However, autophagy-deficient cells are unable to metastasize from the primary tumor due to the inability to disassemble focal adhesions as the result of increased levels of PXN. Reducing PXN levels restores both focal adhesion morphology and motility, demonstrating that the motility defects in autophagy-deficient cells are due to the inability to degrade PXN and thus properly regulate focal adhesion dynamics.

The interaction of cargo destined for autophagic degradation with LC3/Atg8-related molecules at the elongating phagophore is mediated through LIR motifs characterized by the conserved sequence [W/F/Y]-xx-[L/I/V]. We have added to the ever-growing list of LIR-containing proteins with the identification of an evolutionarily conserved LIR motif at residues 40� (YQEI) in the N-terminal region of PXN. In vitro binding assays demonstrated a direct interaction between LC3B and PXN, and the PXN LIR motif is required for LC3B-PXN colocalization and co-immunoprecipitation in metastatic cells. Contrary to other reports, we observed no requirement for the SQSTM1/p62 or NBR1 cargo receptors in PXN degradation.

Given the utility of autophagy for degrading large macromolecular structures and PXN's role as a scaffolding protein within focal adhesions, our work suggests that PXN acts as an autophagy cargo receptor for focal adhesions, forming a bridge between phagophore-bound processed LC3B and focal adhesion proteins. Indeed, other groups have also observed elongating phagophores at focal adhesions. Alternatively, autophagy may siphon PXN from focal adhesions to promote focal adhesion disassembly, although ongoing work in our lab suggests that PXN localization at focal adhesions is required for interaction with LC3B, possibly because of additional direct or receptor-mediated interactions between phagophores and other focal adhesion proteins, including autophagy-regulated proteins such as SRC. Whether autophagic degradation of PXN represents a unique method of regulation in metastatic cells or a general requirement in cell motility remains to be determined.

Interactions of LIR-containing proteins with LC3 can be regulated by post-translational modifications, including phosphorylation both around (e.g., in the cases of OPTN and BNIP3) and within (e.g., in the case of FUNDC1) the LIR motif. The +1 residue of the PXN LIR motif, Y40, is an experimentally validated but previously uncharacterized target of SRC, a known modulator of PXN and focal adhesion turnover. We demonstrated that oncogenic SRC strongly promotes the PXN-LC3B interaction in a manner dependent on Y40 but independent of canonical SRC phosphorylation at Y31 and Y118. In conjunction with observations that the interaction is dependent on SRC kinase activity, these data suggest that phosphorylation of the LIR Y40 residue promotes the PXN-LC3B interaction, in contrast to negative SRC regulation of the FUNDC1-LC3 interaction. Interestingly, we observed that a Y40E phosphomimetic PXN mutant is highly unstable in cells, consistent with increased autophagic turnover of PXN phosphorylated on Y40. However, we cannot exclude the possibility that the SRC effect on focal adhesion disassembly involves the phosphorylation of other sites or proteins. Most intriguingly, the ability of oncogenic SRC to promote migration/invasion was completely abrogated in the absence of intact autophagy, which represents the first suggestion that autophagy may be critical to SRC-mediated motility ( Fig. 1 ).

Novel role for autophagy downstream of oncogenic SRC in promoting focal adhesion disassembly and tumor cell motility. Analyses from primary human cancers and data from genetically engineered mouse models link autophagy to tumor progression to metastasis. However, the molecular mechanisms by which autophagy promotes metastasis are relatively unknown. Our work contributes to a greater understanding of how autophagy promotes metastasis by identifying a critical role for autophagy in metastatic breast and melanoma cells in stimulating focal adhesion disassembly and tumor cell motility. This was achieved through autophagic targeting and degradation of PXN, a critical focal adhesion protein that we show interacts directly with LC3B at the phagophore. Significantly in terms of tumorigenesis, this direct protein-protein interaction between PXN and LC3B is strongly enhanced by oncogenic SRC and, indeed, we also showed that functional autophagy is required for SRC-induced migration and invasion by metastatic tumor cells.

SRC is frequently activated or overexpressed in solid tumors. Although not a primary driver of tumorigenesis, SRC has been suggested to play a critical role in bone metastases in breast cancer. Thus, SRC inhibitors such as dasatinib may represent an effective means of halting metastasis of breast and other cancers, and improved SRC inhibitors with increased selectivity and efficacy are currently being developed. Although clinical trials indicate that SRC inhibitor monotherapy may be ineffective in cancer therapy, our work suggests that autophagy inhibitors may potentiate SRC therapy by targeting a specific requirement for autophagy in SRC-regulated focal adhesion turnover. The potential to prevent tumor cell metastasis could prove to be highly useful within the context of ductal carcinoma in situ (DCIS) and other isolated primary tumors.

Our work has focused on the role of autophagy in focal adhesion turnover in metastatic cells ( Fig. 1 ). There is a growing body of work linking autophagy to different stages of the metastatic cascade, including the secretion of pro-migratory factors, epithelial-to-mesenchymal acquisition, and anoikis resistance. With respect to migration, autophagy exhibits reciprocal regulation of the RHO family of GTPases and cytoskeletal dynamics, and may be involved in the intracellular degradation of extracellular matrix components. As in primary tumor growth, the effect of autophagy on motility is likely to differ among cell types, and our work indicates that SRC status may dictate which cells rely on autophagy for turnover of focal adhesions. It will be interesting to determine whether autophagic focal adhesion turnover and the requirement for intact autophagy in SRC-stimulated motility are required during nonpathological wound healing or immune cell migration. Indeed, autophagy is well-positioned to act as a key regulatory node that allows cells to respond to extracellular or intracellular conditions by balancing cell movement to nutrient-replete areas or metastatic niches with motility arrest, and prosurvival autophagy or cellular death. Future work exploring autophagy in the context of physiological and pathological cell migration will likely reveal many more connections between autophagy and cell motility.

Author information


Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Enders 1309, 320 Longwood Avenue, Boston, 02115, Massachusetts, USA

Dejian Ren, Betsy Navarro, Alexander C. Jackson, Shyuefang Hsu, Qing Shi & David E. Clapham

Massachusetts General Hospital and Department of Obstetrics, Vincent Center for Reproductive Biology, Gynecology and Reproductive Biology, Harvard Medical School, Boston, 02114, Massachusetts, USA

Access options

Get full journal access for 1 year

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Get time limited or full article access on ReadCube.

All prices are NET prices.

Interferon-Inducible Protein 9 (CXCL11)-Induced Cell Motility in Keratinocytes Requires Calcium Flux-Dependent Activation of μ-Calpain

FIG. 1 . IP-9expressed in wounded keratinocytes induces motility of undifferentiatedkeratinocytes (HEKn cells) (a) and immortalized keratinocytes(HaCaT cells) (b) expressing both calpain isoforms (c). (a)Early-passage human keratinocytes were tested for induced motility inan in vitro wound healing assay. Cells were treated with EGF (1 nM) orIP-9 (50 ng/ml) and a pharmacological inhibitor of calpain, calpaininhibitor 1 (5 μM), throughout the assay. The values are shownas the ratio of EGF (1 nM)-induced cell motility. The values are means±standard error of the mean of three independent studies eachperformed in triplicate. Statistical analysis was performed byStudent's t test. (b) Human immortalized keratinocytes,HaCaT cells, were treated with EGF (1 nM) or IP-9 (50 ng/ml)and calpain inhibitor 1 (5 μM) throughout the assay. The valuesare mean ± standard error of the mean of three independentstudies, each performed in triplicate. Statistical analysis wasperformed by Student's t test. (c) Casein zymographydemonstrates that both HEKn and HaCaT keratinocytes possesspotential calpain activity from both ubiquitous isoforms. Proteins(approximately 40 μg) from both HEKn and HaCaTkeratinocytes were electrophoresed into a casein gel and subsequentlyincubated in buffer containing 20 mM MOPS, pH 7.5-5 mM2-mercaptoethanol and calcium (5 mM). Shown here are the Coomassieblue-stained casein gels. Purified porcine M- and μ-calpains (1μg each) served as controls. Images are representative of threeseparate experiments. The data presented in part a are independentlyderived but similar to those publishedbefore(42) the data areprovided herein forcontext. FIG. 2 . IP-9and EGF activate calpain in undifferentiated keratinocytes.Undifferentiated keratinocytes (HEKn cells) (a) and immortalizedkeratinocytes (HaCaT cells) (b) were tested for calpainactivity following stimulation by IP-9 (50 ng/ml) for 120 min or EGF (1nM) for 10 min by intracellular cleavage of the synthetic substrateBoc-LM-CMAC and subsequent fluorescence, as shown by a representativeexperiment of three to five experiments. (a) The calpain inhibitorcalpain inhibitor 1 (5 μg/ml) and the second calpain inhibitorZ-LLY-FMK (10 μM) were added for 30 min prior to the additionof ligand. (c) The involvement of calpain was confirmed by ex vivo MAP2cleavage in HEKn cells as described. Shown is MAP2 fluorescence overunstimulated cells of two experiments performed in triplicate.Statistical analysis was performed by Student's t test.The Boc-LM-CMAC fluorescence is not cell size dependent, and theaddition of the substrate Boc-LM-CMAC to the cells does not change cellsize or shapesignificantly. FIG. 3 . Molecularinterventions downregulate ubiquitous calpains in an isoform-specificmanner. siRNAs targeted against either μ-calpain (a)or M-calpain (b) were transfected into HaCaT cells, and theprotein level of the isoforms was determined 48 h later byisoform-specific immunoblotting. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) staining demonstrates equal cell loading. Shownis one of up to four similar immunoblots for eachanalysis. FIG. 4 . IP-9requires μ-calpain for calpain activity. (a and b)IP-9 activates μ-calpain, whereas EGF triggers M-calpain.Undifferentiated primary keratinocytes (HEKn cells) were treated withIP-9 (50 ng/ml) or/and EGF (1 nM) in the presence or absence ofantisense oligonucleotides (20 μM) to M- and μ-calpainor a scrambled oligonucleotide for 8 h to deplete endogenouscalpain (17). IP-9 andEGF were removed, and the oligonucleotides were replenished for anadditional 12 h. Cells were then again stimulated with IP-9or EGF for 120 min and 10 min, respectively, and calpain cleavage ofthe substrate Boc-LM-CMAC was observed. Representative experimentimages (of three experiments) are shown in a and quantified in b.Values are means ± standard errors of the means of twoindependent studies, each performed in duplicate. Statistical analysiswas performed by Student's t test. (c) siRNAelimination of μ-calpain limits IP-9 induction of BOCfluorescence in HaCaT cells 48 h after transfectionwith isoform-specific siRNA or GFP-targeted siRNA,HaCaT cells were exposed to IP-9 or EGF for 120 min and 10min, respectively, and calpain cleavage of the substrate Boc-LM-CMACwas observed. Representative experiment images (of two experiments) areshown. FIG. 5 . IP-9requires μ-calpain for cell migration and cell de-adhesion. (a)Antisense downregulation of μ-calpain but not M-calpain blocksIP-9-induced motility of HEKn keratinocytes in an in vitro woundhealing assay. Cells were treated in the presence or absence of EGF (1nM) or IP-9 (50 ng/ml) in the presence of antisense oligonucleotidesdirected against the initiation codon regions of M- andμ-calpain or a scrambled oligonucleotide (not shown) for24 h. Cell motility was calculated as a percentage ofEGF-induced responses in the absence of oligonucleotide exposure.Values are means ± standard errors of the means of threeindependent studies, each performed in triplicate. (b and c)siRNA downregulation of μ-calpain but not M-calpainblocks IP-9-induced motility and siRNA downregulation of M-calpain but not μ-calpain blocks EGF induced motility ofHaCaT cells 48 h after transfection, HaCaTcells were exposed to EGF or IP-9 in an in vitro wound healing assay.The values are shown as the ratio of EGF (1 nM)-induced cell motility.Values are means ± standard errors of the means of threeindependent studies, each performed in duplicate. (d) Antisensedownregulation of μ-calpain blocks IP-9 induced de-adhesion andantisense downregulation of M-calpain blocks EGF induced deadhesion inundifferentiated keratinocytes. Cells were treated with EGF (1 nM) andIP-9 (50 ng/ml) and antisense oligonucleotides specific for M-calpainor μ-calpain for 8 h, recovered in the presence ofantisense oligonucleotides for 14 h, and then stimulated withIP-9 or EGF for 2 h and 30 min, respectively. Values arecalculated as a percentage of precentrifugation cells remainingadherent. Values are means ± standard errors of the means oftwo independent studies, each performed in duplicate. Statisticalanalyses were performed with Student's ttest. FIG. 6 . IP-9and EGF induce disassembly of vinculin aggregates. HaCaTkeratinocytes were exposed to EGF or IP-9 for 60 min prior to fixingand staining for vinculin by immunofluorescence (a time coursedemonstrated loss of aggregates starting by 30 min data not shown).One subset of cells was exposed to calpain inhibitor 1 during thefactor exposure. In another series of experiments, the keratinocyteswere treated with isoform-specific siRNA to target M- orμ-calpain independently. Vinculin staining was imaged byconfocal microscopy, and representative cells are shown (threeindependent experiments were performed for each inhibitorychallenge). FIG. 7 . IP-9-inducedFAK cleavage is μ-calpain-dependent. EGF (10 nM) and IP-9 (200ng/ml) stimulation resulted in the appearance of a ≈84-kDacleavage product of FAK as detected by immunoblotting. The appearanceof this fragment was maximal at 30 to 60 min (shown are 30-minchallenges). Concomitant treatment with calpain inhibitor 1 (a) orisoform-specific siRNA (b) reduced the level of this cleavageproduct of FAK. Shown is one of three similar immunoblots for eachsituation. FIG. 8 . Calciumflux elicited by IP-9 was inhibited by intracellular calcium chelatorBAPTA-AM. (a) Intracellular calcium concentration rose in response toIP-9 stimulation in HaCaT cells. The frames shown were taken60 s after addition of IP-9 or diluent (Notx) BAPTA-AM wasadded 40 min prior to IP-9. HEKn cells demonstrated asimilar, BAPTA-AM-quenchable rise in calcium concentration upon IP-9exposure (data not shown). (b) Representative graph of a single celltrace of fluorescence showing average intracellular calcium in controlcells (middle line) and calcium traces in specific cells showingtransients in IP-9-stimulated cells (top line). No calcium fluxes wereobserved when IP-9 was added along with BAPTA-AM (bottomline). The mean intracellular calcium concentration of all IP-9-treatedcells was greater on average from the control cells (see panel a andalso the movie in the supplemental material), but the average rise incalcium concentration is not provided due to expected temporal offsetsof the peaks in intracellular calcium concentration. Shown is arepresentative experiment of three, each done in triplicate, for bothHaCaT and HEKncells. FIG. 9 . Intracellularcalcium is required for IP-9-induced calpain activity. Undifferentiatedkeratinocytes were treated in the presence orabsence of IP-9 (50 ng/ml) for 120 min or EGF (1 nM) for 10min and visualized (a) and quantitated (mean ± standard errorof the mean of >15 cells/experiment) (b) for calpainactivity. BAPTA-AM, a membrane-permeant acetoxymethyl ester of anintracellular calcium chelator, was used to determine the requirementof intracellular calcium for IP-9-induced calpain activity. BAPTA-AM (5μM) was loaded 30 min prior to Boc-LM-CMAC and in the presenceor absence of EGF and IP-9. This experiment was performed three times.Statistical analysis was performed by Student's t test.(c) A similar experiment was performed in human immortalizedkeratinocytes (HaCaT cells) to reproduce the results obtainedwith undifferentiatedkeratinocytes. FIG. 10 . ERKis required for EGF-induced cell migration. a) EGF (1 nM, 10 min) butnot IP-9 (50 ng/ml, 120 min) induces ERK MAP kinase in undifferentiatedkeratinocytes. Forskolin (25 μM, 15 min prior to ligand) servedas an independent activator of cyclic AMP. Activation of ERK wasdetermined by immunoblotting equal protein lysates for phospho-ERK. (b)Pharmacological inhibition of MAP kinase kinase with PD98059 (2μM, 30 min prior to ligand) prevents EGF-induced calpaincleavage of the Boc-LM-CMAC substrate. Images are representative ofthree experiments. (c) PD98059 (2 μm) blocked cell migration inundifferentiated keratinocytes induced by EGF (1 nM) but not IP-9 (50ng/ml) (n = 3). Statistical analysis was performed byStudent's ttest. FIG. 11 . IP-9-inducedcell migration requires PLC-dependent calpain activation. (a)Pharmacological inhibition of all PLC isoforms by U-73122 (2μM) blocked both EGF- and IP-9-induced cell motility inundifferentiated keratinocytes. The values are shown as ratios of theEGF (1 nM)-induced cell motility. Thevalues are means ± standard errors of the means of sixindependent studies, each performed in triplicate.Statistical analysis was performed by Student'st test. (b) Undifferentiated keratinocytes were tested forcalpain activity following stimulation by IP-9 (50 ng/ml) for 120 minor EGF (1 nM) for 10 min. The pan-PLC inhibitor ET-18-OCH3 (100 nM) andthe phospholipase D inhibitor propranolol (100 μM) (as anonspecific control in addition to molecular downregulation) were addedfor 30 min prior to the addition of ligand. Calpain activity wasmonitored by intracellular cleavage of the syntheticsubstrate Boc-LM-CMAC and subsequent fluorescence. Shown isa representative experiment of three to fiveexperiments. FIG. 12 . PLC-β3signaling is required for IP-9 induced calpain activation and motility.Undifferentiated keratinocytes were grown in the presence orabsence of antisense oligonucleotides (20 μM) to PLC-β3(or PLC-γ1) for 48 h in quiescence medium. After thistime, cells were stimulated with IP-9 (50 ng/ml) or EGF (1nM) for 120 min or 10 min, respectively, and calpain cleavage of thesubstrate Boc-LM-CMAC was ob served. Representativeexperiment images (of three experiments) are shown (a) and quantified(mean ± standard error of the mean of >15cells/experiment) (b). Statistical analysis was performed byStudent's t test. (c) PLC-β3 levels were assessedby immunoblotting cells exposed to specific (antisense PLC-β3)or irrelevant (trnfx control) oligonucleotides. Shown is arepresentative of at least three such immunoblots. trnfx,transfection.

Materials and methods

Algal culture and experimental setup

For the experiments, cultures of the unicellular photosynthetic freshwater flagellate Chlamydomonas nivalis (F. A. Bauer) Wille 1903 are used. They are grown by inoculating 1 ml of a logarithmic phase culture into 50 ml of Bold's basal medium + 5% sterilized soil extract. The culture is kept at 23.4°C under a light of about 6000 lux=8.8 W m –-2 from cool, white-tone fluorescent lights turned on for 16 h a day. Measurements are performed 2 weeks after the inoculation of the culture. Microscopic observation shows that the diameter of an individual cell in these cultures lies in the range 3–5 μm.

The experimental setup used is generally similar to the one described in Vladimirov et al. (2000), see Fig. 1A. An argon ion laser with a wavelength of 514 nm (green) and light intensity of 1400 W m –2 is the only light source in the experiments. Laser light passes through a cylindrical lens and a diaphragm, which produces a vertically oriented light sheet with a cross-section of 14 mm×1.5 mm. The laser sheet is directed along the plane of symmetry of the vertically positioned test tube of rectangular cross-section(Fig. 1B) containing medium and algal cells. A mirror is placed behind the test tube to make the cells'illumination symmetric, thus avoiding bias in their self-swimming. Images are acquired with a Kodak Megaplus 1.4 CCD camera, resolution of 1316×1034 pixels 2 . The laser, optical system and acquisition system belong to a Particle Image Velocimetry (PIV) System (TSI Inc., Shoreview, MN, USA).

Motility, the ability of an organism to move independently, using metabolic energy, [2] [3] can be contrasted with sessility, the state of organisms that do not possess a means of self-locomotion and are normally immobile. Motility differs from mobility, the ability of an object to be moved. The term vagility encompasses both motility and mobility sessile organisms including plants and fungi often have vagile parts such as fruits, seeds, or spores which may be dispersed by other agents such as wind, water, or other organisms. [4]

Motility is genetically determined, [5] but may be affected by environmental factors such as toxins. The nervous system and musculoskeletal system provide the majority of mammalian motility. [6] [7] [8]

In addition to animal locomotion, most animals are motile, though some are vagile, described as having passive locomotion. Many bacteria and other microorganisms, and multicellular organisms are motile some mechanisms of fluid flow in multicellular organs and tissue are also considered instances of motility, as with gastrointestinal motility. Motile marine animals are commonly called free-swimming, [9] [10] [11] and motile non-parasitic organisms are called free-living. [12]

Motility includes an organism's ability to move food through its digestive tract. There are two types of intestinal motility – peristalsis and segmentation. [13] This motility is brought about by the contraction of smooth muscles in the gastrointestinal tract which mix the luminal contents with various secretions (segmentation) and move contents through the digestive tract from the mouth to the anus (peristalsis). [14]

At the cellular level, different modes of movement exist:

    , a swimming-like motion (observed for example in spermatozoa, propelled by the regular beat of their flagellum, or the E. coli bacterium, which swims by rotating a helical prokaryotic flagellum) , a crawling-like movement, which also makes swimming possible [16][17] , a form of motility used by bacteria to crawl over surfaces using grappling hook-like filaments called type IV pili. , enabling movement of the axonalgrowth cone[18]

Many cells are not motile, for example Klebsiella pneumoniae and Shigella, or under specific circumstances such as Yersinia pestis at 37 °C. [ citation needed ]

Events perceived as movements can be directed:

  • along a chemical gradient (see chemotaxis)
  • along a temperature gradient (see thermotaxis)
  • along a light gradient (see phototaxis)
  • along a magnetic field line (see magnetotaxis)
  • along an electric field (see galvanotaxis)
  • along the direction of the gravitational force (see gravitaxis)
  • along a rigidity gradient (see durotaxis)
  • along a gradient of cell adhesion sites (see haptotaxis)
  • along other cells or biopolymers

Muscles give the ability for voluntary movement, and involuntary movement as in muscle spasms and reflexes). At the level of the muscular system, motility is a synonym for locomotion. [19] [20]

Most sperm have a single flagellum to help them swim. The cervical, uterine, and fallopian linings of the female reproductive system play a more important role in transporting sperm to ova.

The record speeds cheetahs hold are owed in large to their muscle motility.

The shoots of plants move by growing towards light. This is known as positive phototropism. The roots grow away from light. This is known as negative phototropism.

Monocytes and macrophages of the immune system engulf Bacteria by extending their pseudopodia. Note that this cartoon is not an accurate representation of phagocytosis.

Motility at the sub-cellular level. This depicts translation - a motile nanoscale molecular process using protein dynamics.

Watch the video: Cytoskel Actin Cell motility (May 2022).


  1. Sewell

    We must have a look !!!

  2. Templeton

    The question is interesting, I too will take part in discussion.

  3. Pearroc

    They are wrong. Let us try to discuss this. Write to me in PM, speak.

  4. Amoll

    well ...... test !!!

Write a message