Optogenetic dissection of mitotic spindle positioning in vivo
Animal cells control the position of the spindle to determine the plane of cell cleavage . Regulated spindle positioning is therefore critical for asymmetric cell division and tissue formation ( di Pietro et al . , 2016 ) . Early work in C . elegans demonstrated that cortical pulling forces position the spindle through a protein complex that consists of a heterotrimeric G protein alpha subunit , GOA-1Gαo or GPA-16Gαi ( together referred to as Gα ) , a TPR-GoLoco domain protein GPR-1/2 , and the coiled-coil protein LIN-5 ( Colombo et al . , 2003; Gotta and Ahringer , 2001; Gotta et al . , 2003; Grill et al . , 2001; Lorson et al . , 2000; Miller and Rand , 2000; Srinivasan et al . , 2003 ) . This complex , and the closely related Drosophila Gαi/o–Pins–Mud and mammalian Gαi/o–LGN–NuMA protein complexes , recruit the microtubule motor dynein to the cell cortex ( Bellaïche et al . , 2001; Bowman et al . , 2006; Du and Macara , 2004; Du et al . , 2001; Izumi et al . , 2004; Nguyen-Ngoc et al . , 2007; Schaefer et al . , 2001; Zheng et al . , 2010; Zhu et al . , 2011 ) ( Figure 1a ) . While regulation at the level of individual components has been described , it remains unclear whether these proteins only form a physical anchor for dynein , or whether individual subunits contribute additional functions in spindle positioning . As a potential additional function , force generation may require a dynein adaptor that activates dynein motility . Such an adaptor is necessary for the processive movement of mammalian cytoplasmic dynein during cargo transport along microtubules ( Reck-Peterson et al . , 2018 ) . This process differs substantially from microtubule-dependent cortical pulling , in which force is generated by dynein in association with shrinking microtubules ( Laan et al . , 2012 ) . Without adaptor , surface-anchored yeast dynein in contact with depolymerizing microtubules generates pulling forces in vitro ( Laan et al . , 2012 ) . However , yeast dynein moves processively on its own ( Reck-Peterson et al . , 2006 ) . Hence , it remains unknown whether cortical pulling force generation in animal cells depends just on anchoring of dynein , or whether this requires an additional dynein activator . The role of Gαi/o subunits in pulling force generation has also remained ambiguous ( Figure 1a ) . Membrane-attached Gα∙GDP associates with GoLoco motifs present in the homologous GPR-1/2 , Pins , and LGN proteins ( Kimple et al . , 2002; Schaefer et al . , 2001 ) . This preference for the GDP-bound ‘inactive’ Gα state explains why RGS-7 , a putative GTPase activating protein ( GAP ) , promotes spindle positioning ( Hess et al . , 2004 ) . However , the role of another conserved regulator of Gα signaling , RIC-8 , remains poorly understood ( Afshar et al . , 2004; Miller and Rand , 2000; Tall et al . , 2003 ) . RIC-8 was shown to act as a guanine nucleotide exchange factor ( GEF ) in vitro , while it may function in vivo as a Gα chaperone or as both a GEF and chaperone ( Afshar et al . , 2004; Afshar et al . , 2005; David et al . , 2005; Gabay et al . , 2011; Hampoelz et al . , 2005; Tall et al . , 2003; Wang et al . , 2005 ) . In addition to RIC-8 , G-protein coupled receptors and Gαo∙GTP signaling contribute to spindle positioning in Drosophila neuroblasts and sensory organ precursor cells ( Katanaev et al . , 2005; Schaefer et al . , 2001; Yoshiura et al . , 2012 ) . Therefore , it has been proposed that the Gα∙GTP-binding and hydrolysis cycle forms a critical step in cortical pulling force generation ( Afshar et al . , 2004; Srinivasan et al . , 2003; Yoshiura et al . , 2012 ) . However , it is difficult to distinguish whether Gαo∙GTP contributes to force generation , or more indirectly relays cell-cell signaling to the spindle . Here , we describe an optogenetic strategy for the systematic examination of individual contributions of cortical pulling force components in vivo . We use the C . elegans one-cell embryo ( P0 ) , which undergoes reproducible spindle positioning and asymmetric cell division in the absence of cell-cell signaling ( Video 1 ) ( Rose and Gonczy , 2014 ) . As an initial hurdle , modifying endogenous genes with tunable light-controlled interacting protein tags ( TULIPs ) induced strong germline silencing . We developed a strategy to promote expression of foreign sequences in the C . elegans germline , which is based in part on a new codon usage adaptation method ( GLO , GermLine Optimized ) . This allowed the light-controlled localization of endogenous proteins through ePDZ–LOV domain interactions in the early C . elegans embryo . Our results show that Gα∙GDP and GPR-1/2 can be replaced with a light-inducible membrane anchor , while LIN-5 is required as activator of dynein-dependent cortical pulling force generation . Local light-controlled LIN-5 recruitment enabled us to manipulate the spindle position and orientation , and thereby the outcome of cell division in the early embryo .
We set out to systematically investigate the individual roles of the proteins involved in cortical pulling force generation . Our previous studies and CRISPR/Cas9-assisted endogenous tagging demonstrated that cytoplasmic dynein and the Gα–GPR-1/2–LIN-5 complex overlap and function together in pulling force generation at the cell cortex of C . elegans early blastomeres ( Figure 1b ) ( Portegijs et al . , 2016; Schmidt et al . , 2017; van der Voet et al . , 2009 ) . As a read-out for pulling forces , we measured spindle pole peak velocities after UV-laser ablation of the spindle midzone ( Grill et al . , 2001 ) ( Figure 1c and Video 2 ) . Interfering with Gα , GPR-1/2 , or LIN-5 function abolished significant force generation , as previously reported ( Figure 1d ) . RNA interference ( RNAi ) of ric-8 or rgs-7 by dsRNA injection resulted in partial loss of pulling forces ( Figure 1e ) . Double ric-8 ( RNAi ) ; rgs-7 ( RNAi ) did not further decrease pulling forces as might be expected when RIC-8 and RGS-7 both promote a critical GTPase cycle ( Hess et al . , 2004; Srinivasan et al . , 2003 ) . However , RNAi of ric-8 and rgs-7 is known to cause incomplete gene inactivation , which could also explain the limited defects . To circumvent this caveat , we set out to generate germline-inducible knock-out alleles , as ric-8 and rgs-7 null mutants produce no or very few viable progeny ( Hess et al . , 2004; Reynolds et al . , 2005 ) . To this end , we inserted lox sites in the endogenous ric-8 and rgs-7 loci by CRISPR/Cas9-assisted recombineering ( Figure 1—figure supplement 1 ) , and expressed the CRE recombinase specifically in the germline ( Figure 1—figure supplement 1 ) . Compared to the control without CRE activity , knockout embryos showed reduced spindle pole peak velocities ( ric-8lox: anterior −50% and posterior −48%; rgs-7lox: anterior −29% and posterior −11% ) , similar to or more defective than the corresponding RNAi embryos ( Figure 1e and Figure 1—figure supplement 2 ) . Importantly , the double knock-out of ric-8lox; rgs-7lox showed much reduced spindle pole movements ( anterior −68% and posterior −67% ) , thereby resembling Gα ( RNAi ) ( Figure 1e ) . This indicates that RIC-8 and RGS-7 act independently , or partly redundant , as positive regulators of Gα . To gain further insight into the individual functions of cortical pulling force regulators , we sought to obtain spatiotemporal control of protein localization . To this end , we explored implementing the ePDZ–LOV system , which makes use of exposure to blue light to control protein heterodimerization ( Harterink et al . , 2016; Strickland et al . , 2012 ) . As introduction of epdz , lov , and cre sequences induced strong germline silencing responses , we developed a computational approach to design protein-coding sequences that are resistant to silencing in the germline . Our design algorithm assembles a coding sequence for any desired polypeptide from a list of 12-nucleotide words found in native germline-expressed genes ( Figure 2—figure supplement 1 ) . We hypothesized that transgenes designed in this way would mimic native genes and thereby evade the germline silencing machinery . Indeed , using this approach , we could obtain robust expression of several foreign transgenes that were otherwise silenced ( Figure 2—figure supplement 2 ) . Although most of these transgenes were stably expressed for many generations , two out of 16 distinct constructs tested showed evidence of gradual silencing when passaging strains in culture ( Figure 2—figure supplement 3 ) . Therefore , as a further buffer against silencing , we combined our germline-optimized exons with poly-AT-cluster rich intron sequences , which were recently demonstrated to protect against germline silencing ( Frøkjær-Jensen et al . , 2016; Zhang et al . , 2018 ) . This combined approach resulted in stable germline expression of eight out of eight transgenes and enabled implementation of the ePDZ–LOV system for use in the C . elegans early embryo . To characterize the ePDZ–LOV system , we created a strain with a membrane-bound LOV2 domain , expressed as a pleckstrin-homology domain ( PH ) –eGFP protein fusion ( PH::LOV ) , together with cytosolic ePDZ::mCherry ( Figure 2a ) . Illumination with a blue ( 491 nm ) laser rapidly induced recruitment of ePDZ::mCherry to PH::LOV , and allowed both global and local cortical enrichment in one-cell embryos ( Figure 2b and Videos 3–5 ) . Because GFP is also excited with blue light , experiments that involve GFP imaging imply global and continuous induction of the ePDZ–LOV interaction . To test the reversibility of the ePDZ–LOV interaction , we followed ePDZ::mCherry membrane localization after a global activation pulse , and found dissociation kinetics similar to those reported by others ( Hallett et al . , 2016 ) ( t½=42 s; Figure 2c and Video 6 ) . Thus , we conclude that the ePDZ–LOV system is suitable for controlled protein localization in the early C . elegans embryo . Next , we examined whether membrane recruitment of RGS-7 and RIC-8 promotes pulling forces . This could provide insight into the in vivo relevance of the Gα∙GDP/GTP cycle and indicate whether RIC-8 is more likely to contribute as a chaperone or as a GEF . Regulation of the Gα∙GDP/GTP cycle normally takes place at the cell membrane , while chaperoning of Gα folding and trafficking is expected to occur in the cytosol and at endomembranes ( Gabay et al . , 2011 ) . We created strains expressing endogenous RIC-8 and RGS-7 as ePDZ::mCherry protein fusions . When combined with PH::LOV , this resulted in light-inducible membrane recruitment of RIC-8 and RGS-7 ( Figures 3a and 4a , and Video 7 ) . Global cortical enrichment of RGS-7 enhanced spindle pole movements ( anterior +25% and posterior +20% ) ( Figure 3b ) and spindle oscillations ( Figure 3c ) . The RGS-7::ePDZ signal was too subtle to reliably control its local recruitment . As an alternative strategy , we fused eGFP::LOV to endogenous PAR-6 , which localizes to the anterior cortex of the zygote ( Figure 3a ) . Following global light exposure , recruitment of RGS-7::ePDZ to PAR-6::LOV enhanced the peak velocities of both spindle poles , but most significantly the movement of the anterior pole ( anterior +25% and posterior +14%; Figure 3b , c ) . Thus , cortical recruitment of RGS-7 acutely increases pulling forces , in agreement with its proposed function as a GAP that promotes Gα∙GDP–GPR-1/2 interaction . In contrast , cortical enrichment of RIC-8 did not significantly enhance pulling forces ( Figure 4b ) . Thus , in agreement with the ric-8lox; rgs-7lox synergistic phenotype , our optogenetic localization experiments support a model in which RIC-8 and RGS-7 promote Gα function at different levels . While RGS-7 probably functions as a GAP , our data are in line with RIC-8 acting in vivo as a Gα chaperone , rather than a GEF , thus indirectly promoting force generation . To directly address whether Gα∙GTP might contribute to spindle positioning and if Gα∙GDP serves merely as a membrane anchor , we aimed to reconstruct a cortical force generator in the absence of Gα ( Figure 5a ) . We obtained optogenetic control over the membrane localization of GPR-1 by combining endogenously labeled epdz::mcherry::gpr-1 ( ePDZ::GPR-1 ) with knockout of gpr-2 , expression of PH::LOV and Gα RNAi ( Figure 5b , c and Videos 8 and 9 ) . Live imaging and immunohistochemistry confirmed light-induced cortical recruitment of ePDZ::GPR-1 and consequently LIN-5 ( Figure 5b and Figure 5—figure supplement 1 ) . Spindle movements appeared reduced following the tagging of gpr-1 and knockout of gpr-2 ( anterior −40% and posterior −26% ) ( Figure 5—figure supplement 2 ) . However , light-induced ePDZ::GPR-1 recruitment increased spindle pole movements ( anterior +56% and posterior +10% ) ( Figure 5d , f ) . Moreover , membrane-localized ePDZ::GPR-1 sustained force generation in the absence of Gα ( anterior +195% and posterior +232% ) , indicating that Gα is dispensable for cortical pulling force generation . Recruitment of ePDZ::GPR-1 restored spindle pole movements to a similar degree in Gα ( RNAi ) and Gα ( RNAi ) ; ric-8 ( RNAi ) embryos ( Figure 5d ) . Thus , cortical pulling forces can be generated when the Gα membrane anchor function is replaced by PH::LOV , and most likely in the absence of Gαi/o∙GTP . We conclude that Gα functions as a membrane anchor and that Gα∙GTP does not perform an essential function in pulling force generation . Light-controlled heterodimerization exhibits a certain level of dark state activity ( Hallett et al . , 2016 ) . We performed spindle severing experiments in the absence of blue light to confirm the light-specificity of recruitment . Replacement of gfp::tubulin with an mcherry::tubulin transgene allowed for tracking of the spindle in the absence of blue light and consequently LOV activation . We observed that the scattering of UV-light ( 355 nm ) during midzone ablation also uncages the LOV domain . Nevertheless , the presence of blue light resulted in substantially elevated spindle pulling forces when compared to dark state experiments ( anterior +33% and posterior +35% ) ( Figure 5e ) . We conclude that the observed spindle pole movements are light-dependent and the specific result of inducible cortical recruitment of GPR-1 . Considering that spindle poles moved in three dimensions after recruitment of GPR-1 , measuring peak velocities after midzone severing by tracking the poles in two dimensions likely underestimates the resulting pulling forces . Therefore , we used an additional read-out of cortical pulling forces . Cortical pulling events cause inward movements of the plasma membrane , which are visible by spinning disk-confocal fluorescence microscopy as extended invaginations of the plasma membrane ( Redemann et al . , 2010 ) ( Figure 5g and Videos 10 and 11 ) . These membrane invaginations occur in wild type embryos , depend on microtubules and cortical force generator components , and correlate with the distribution of pulling force generators ( Redemann et al . , 2010 ) . Therefore , these membrane invaginations most likely reveal the presence and distribution of active individual force generators . Using spinning disk confocal microscopy , we quantified membrane invaginations by counting the number of transient cortical PH::GFP dots in the sub-cortical plane . Control PH::GFP embryos showed on average 138 membrane invaginations during anaphase in an area covering approximately 1/3 of the cell surface ( Figure 5g ) . When plotted along the anterior-posterior axis , the distribution of these invaginations reflected the three described cortical domains: anterior , posterior , and a posterior lateral region at ±60% embryo length ( Rose and Gonczy , 2014 ) ( Figure 5h-left ) . The posterior lateral band region localizes the LET-99 DEP-domain protein , which antagonizes the localization of GPR-1/2 and thereby pulling force generation ( Krueger et al . , 2010; Tsou et al . , 2003 ) . This explains the absence of invaginations around 60% embryo length ( Figure 5h-left ) . Cortical GPR-1 recruitment resulted in a total number of 174 ( +25% compared to ph::lov control ) invaginations in the presence , and 122 invaginations ( +249% compared to Gα ( RNAi ) embryos ) in the absence of Gα ( Figure 5 and Figure 5—figure supplement 3 ) . Thus , in agreement with our observations in spindle severing assays , GPR-1 recruitment to the membrane induces cortical pulling forces , even in the apparent absence of Gα proteins . The lack of invaginations around 60% embryo length was no longer detected when ePDZ::GPR-1 was recruited to PH::LOV . In agreement , the characteristic dip in cortical GPR-1 localization ( e . g . : Figure 1b ) was no longer detected after ectopic GPR-1 recruitment ( Figure 5b ) . Thus , as expected , LET-99 does not antagonize the cortical recruitment of ePDZ::GPR-1 by PH::LOV , in contrast to the Gα∙GDP-mediated localization of endogenous GPR-1/2 . The pattern of invaginations still showed two peaks and a mild dip at 50% embryo length ( Figure 5h ) . The remaining peak numbers of invaginations likely represent the cortical regions closest to the spindle poles , as these sites contact the highest numbers of astral microtubules . Taken together , Gα is not essential for force generation , but the characteristic distribution of force generating events is likely regulated in part at the Gα protein or Gα–GPR-1/2 protein interaction level . The Gα–GPR-1/2–LIN-5 complex has been suggested to function as a dynein anchor ( Kotak et al . , 2012; di Pietro et al . , 2016 ) . Our optogenetic approach allows replacing the entire complex by PH::LOV , and examining whether the complex strictly acts as an anchor , or whether individual components have additional functions ( Figure 6a ) . To directly recruit dynein to the cortex , we generated an epdz::mcherry knock-in allele of dhc-1 ( dynein heavy chain ) . While homozygous epdz::mcherry::dhc-1 ( ePDZ::DHC-1 ) was viable , its combination with ph::egfp::lov was lethal . This effect was also observed for an ePDZ::GFP fusion of DHC-1 in the presence of PH::LOV , but not in the absence of PH::LOV or for mCherry::DHC-1 without the ePDZ domain . Therefore , we attributed the lethality to ePDZ–LOV dark state interactions that disturb essential dynein functions . We circumvented this effect by using epdz::mcherry::dhc-1 in combination with a wild-type allele ( epdz::mcherry::dhc-1/+ ) to control dynein localization links to different figure ( Figure 6b , c and Videos 12 and 13 ) . We found that induced ePDZ::DHC-1 cortical recruitment in the presence of the wild type complex slightly ( but not significantly ) increased spindle pole movements ( anterior +10% and posterior +5% , 162 membrane invaginations:+17%; Figure 6d–f ) . In addition , cortical recruitment of ePDZ::DHC-1 slightly increased the nearly absent spindle pole movements in lin-5 ( RNAi ) embryos ( Figure 6—figure supplement 2 ) . Notably , however , cortical ePDZ::DHC-1 recruitment in the absence of a wild type complex ( Gα ( RNAi ) , gpr-1/2 ( RNAi ) or lin-5 ( RNAi ) embryos ) did not result in substantial pulling force generation , spindle movements , or membrane invaginations ( Figure 6d–f and Figure 6—figure supplement 1 ) . Because direct cortical dynein anchoring does not support force generation , it is likely that the LIN-5 complex performs essential functions beyond providing a structural dynein anchor . In vitro reconstitution studies established that homodimerizing adapters containing extended coiled-coil domains are critical for dynein activity ( McKenney et al . , 2014; Schlager et al . , 2014 ) . LIN-5 and its homologs NuMA and Mud are predicted to contain a long coiled-coil domain , to homodimerize , and to interact with dynein ( Kotak et al . , 2012; Lorson et al . , 2000; Merdes et al . , 1996 ) . To investigate if LIN-5 can activate dynein-dependent force generation , we recruited endogenous LIN-5 to the cortex ( Figure 7a–c and Videos 14 and 15 ) . Spindle severing experiments and invagination counting revealed that cortical LIN-5 recruitment greatly increased spindle pulling forces in otherwise wild type embryos ( anterior +131% and posterior +68% , 557 invaginations:+303% ) ( Figure 7d , e and Figure 7—figure supplement 1 ) . gpr-1/2 ( RNAi ) embryos also showed strong dynein-dependent forces after cortical recruitment of LIN-5 ( anterior +183% and posterior +244% , 429 invaginations:+1488% ) . In fact , cortical LIN-5 recruitment generated extreme premature pulling forces ( Video 16 ) that could result in separation of centrosomes and their associated pronuclei even before formation of a bipolar spindle ( Video 17 ) . Therefore , embryos were kept in the absence of blue light until mitotic metaphase . Subsequent blue light exposure induced cortical LIN-5 recruitment within seconds , and the spindles showed excessive movements in all three dimensions well before cortical LIN-5 reached peak levels ( Figure 7c , e , Videos 14 and 15 ) . Therefore , the number of membrane invaginations in anaphase probably reflects the pulling forces more accurately than the average peak velocities of the poles ( Figure 7f and Figure 7—figure supplement 1 ) . These results identify LIN-5 as a strong activator of dynein in the generation of cortical pulling forces . We observed similarly high pulling forces upon cortical LIN-5 recruitment in gpr-1/2 ( RNAi ) and Gα ( RNAi ) embryos ( Figure 7—figure supplement 1 ) . Thus , neither Gα nor GPR-1/2 are required for force generation , and this particular context did not reveal a positive contribution of Gα∙GTP either . In immunofluorescence staining experiments , we observed that cortical recruitment of LIN-5 localized dynein to the cortex ( Figure 7—figure supplement 2 ) . Notably , the reverse was also seen: we detected LIN-5 at the cortex following the direct recruitment of ePDZ::DHC-1 to PH::LOV , even after knockdown of gpr-1/2 by RNAi ( Figure 6—figure supplement 2 , top ) . The p150 ( Glued ) dynactin subunit DNC-1 was also present at the cell cortex of such embryos , which indicates that at least some PH::LOV-localized dynein complexes contain the dynactin cofactor ( Figure 6—figure supplement 2 , lower panels ) . DNC-1p150 colocalized with PH::LOV membrane-recruited dynein even in lin-5 ( RNAi ) embryos . Thus , the cortical localization of dynein through a direct PH::LOV interaction leads to co-recruitment of LIN-5 and dynactin , but not to significant force generation . It is possible that these complexes adapt an inactive conformation or lack specific subunits of the dynein-dynactin motor complex . In contrast , dynein anchored at the cell cortex through the LIN-5 intermediate generated strong pulling forces . Together , these data indicate that association with membrane-attached LIN-5 is essential for dynein to generate cortical pulling forces . Next , we examined whether we could deploy cortical LIN-5 to manipulate spindle positioning and the outcome of cell division by local illumination with blue light . In the normal P0 cell , the spindle is displaced towards the posterior and cell cleavage creates a larger anterior blastomere ( AB ) and a smaller posterior blastomere ( P1 ) . Local recruitment of LIN-5 to the anterior cortex from metaphase onwards caused the P0 spindle to position anteriorly , which inverted the AB:P1 size asymmetry after cell division ( Figure 8a and Video 18 ) . In addition , recruiting LIN-5 laterally induced a completely perpendicular spindle position ( Figure 8b and Video 19 ) . While this triggered some furrowing at the anterior and posterior cell poles , the spindle switched back to an anterior-posterior orientation during cytokinesis , possibly resulting from geometric constraints . We therefore switched to two-cell embryos with the relatively round AB and P1 blastomeres . In two-cell gpr-1/2 ( RNAi ) embryos , the spindle fails to rotate in P1 , resulting in a transverse spindle orientation in both blastomeres ( Srinivasan et al . , 2003 ) . Importantly , local recruitment of LIN-5 to the membranes between AB and P1 promoted anterior-posterior spindle orientations in both blastomeres of gpr-1/2 ( RNAi ) embryos ( Figure 8c and Videos 20 and 21 ) . These spindles maintained their anterior-posterior orientation throughout mitosis and induced cleavage furrows that reproducibly followed the spindle position . These experiments underline the determining role of LIN-5-dependent cortical pulling in spindle orientation and cell cleavage plane determination .
Recent advances in CRISPR/Cas9-mediated genome engineering and optogenetics hold far-reaching potential for cell and developmental biology ( Johnson and Toettcher , 2018; Waaijers and Boxem , 2014 ) . We combined these strategies to systematically control the localization of endogenous proteins in the C . elegans early embryo by light-induced ePDZ–LOV heterodimerization , to determine their individual contributions in spindle positioning . This demonstrated that LIN-5 , but not Gα , RIC-8 and GPR-1/2 , is intrinsically required for dynein-dependent pulling force generation . Based on our quantitative analyses , we propose that Gα–GPR-1/2 provides a regulatable membrane anchor , while membrane-bound LIN-5 acts as an obligate adapter and activator of cytoplasmic dynein at the cell cortex . Our observations fit with and expand on those from studies in which the LIN-5-related Mud and NuMA proteins where tethered to the cortex . In two earlier studies , this resulted in the conclusion that the NuMA complex acts as a dynein anchor , but substitution of components or Gα protein removal was not tested ( Kotak et al . , 2012; Ségalen et al . , 2010 ) . However , a very recent study using human carcinoma cells followed an optogenetic strategy similar to ours , and also observed that a membrane anchor cannot replace the entire Gα–LGN–NuMA complex ( Okumura et al . , 2018 ) . As in C . elegans embryos , a CAAX membrane anchor could substitute for Gα–LGN in human carcinoma cells , while dynein needed to be anchored through NuMA in order to generate spindle positioning forces ( Okumura et al . , 2018 ) . Thus , observations in two different systems indicate that dynein activation at microtubule plus ends requires the LIN-5/NuMA adaptor protein , similar to the requirement for an activating dynein adaptor in cargo transport ( McKenney et al . , 2014; Schlager et al . , 2014; Zhang et al . , 2017 ) . It is tempting to speculate that activation by LIN-5NuMA specifically promotes dynein function in generating membrane associated pulling forces . In the cytoplasm , dynein adopts an inactive conformation and does not show processive movement in the absence of an activating adaptor ( Reck-Peterson et al . , 2018 ) . Adaptor proteins such as BicD2 and Hook3 interact with specific cargo as well as with the dynein tail regions and the universal activating complex dynactin ( McKenney et al . , 2014; Schlager et al . , 2014; Zhang et al . , 2017 ) . Thereby , these adaptors promote the formation of stable and active dynein–dynactin-adaptor complexes . Recent stuctural analyses revealed that the coiled-coil regions of the adaptors extend in between dynein and dynactin , with multiple adaptor-specific interaction sites , and with several adaptors promoting the recruitment of two dynein complexes in parallel ( Grotjahn et al . , 2018; Sladewski et al . , 2018; Urnavicius et al . , 2018 ) . Although structural studies have focused on factors promoting minus-end directed transport , it is likely that dynein-dependent cortical pulling forces also involve an activating adaptor . As such , LIN-5NuMA could promote the assembly of stable dynein–dynactin complexes and release dynein autoinhibition . It is remarkable , however , that direct recruitment of dynein to the cortex did not result in cortical pulling forces , despite the co-recruitment of LIN-5 and the dynactin subunit DNC-1p150 . At present , we do not know whether this reflects individual dynein–LIN-5 and dynein–dynactin interactions , or the formation of stable dynein–dynactin–LIN-5 complexes . The lack of pulling forces , despite co-recruitment of LIN-5NuMA and DNC-1p150 together with dynein , may indicate that the complex needs to be anchored through LIN-5 in order to achieve a specific conformation and facilitate microtubule end-on pulling forces . Such a requirement could prevent inappropriate activation of dynein–dynactin–LIN-5NuMA complexes in the cytoplasm . In the optogenetic experiments in human carcinoma cells , dynein also localized p150 at the membrane , however in contrast to our findings , NuMA was not observed to be co-recruited in this study ( Okumura et al . , 2018 ) . While the reason for this discrepancy is currently unknown , it might be related to the remarkably different kinetics of the two systems . Using the TULIP system , we observed membrane localization of ePDZ-tagged endogenous proteins within seconds , whereas in the iLID experiments in human carcinoma cells accumulation took place over multiple minutes . It is possible that in the longer time frame , cytoplasmic dynein complexes are recruited to the cortex with adaptors other than NuMA . Since only LIN-5 is strictly required for cortical pulling force generation , the question arises why a tripartite dynein anchor is conserved from worm to man . In yeast , dynein is localized by the single-component cortical anchor Num1 , a coiled-coil domain protein with a PH-domain for membrane localization ( Ananthanarayanan , 2016 ) . Our ectopic ePDZ–LOV heterodimerization experiments show that membrane-tethered LIN-5 could suffice as a dynein anchor and activator , and that local regulation is needed to rotate and displace the spindle . Conceivably , the trimeric dynein anchor/adaptor evolved in metazoans to augment context-specific regulation and reduce stochastic activation of spindle pulling forces . Which factors may normally control the Gα–GPR-1/2 membrane anchor ? The LET-99 protein was previously reported to restrict the localization of GPR-1/2 in a posterior-lateral band region of the one-cell embryo , thereby contributing to higher net pulling forces in the posterior direction ( Tsou et al . , 2002; Tsou et al . , 2003 ) . Our observation that direct membrane recruitment of GPR-1 overcomes this regulation is in agreement with LET-99 normally antagonizing Gα∙GDP–GPR-1/2 interaction . Another level of Gα–GPR-1/2 regulation that remains incompletely understood is the contribution of RIC-8 in spindle positioning . The ric-8 gene was discovered through ‘resistant to inhibitors of cholinesterase 8’ mutants in C . elegans , which are defective in Gαq-stimulated neurotransmitter release ( Miller et al . , 2000 ) . The discovery that ric-8 acts also with GOA-1 in spindle positioning indicated a general role for RIC-8 in Gα regulation ( Miller and Rand , 2000 ) . Indeed , two different general functions have been reported . Based on in vitro experiments , mammalian RIC-8A acts as a non-receptor GEF , which surprisingly does not activate Gαβγ trimers , but shows higher affinity for free Gα∙GDP and the Gα∙GDP–LGN–NuMA complex ( Tall and Gilman , 2005; Tall et al . , 2003 ) . In contrast , experiments in Drosophila and mouse embryonic stem cells demonstrated a chaperone function required for the cortical localization of Gα subunits ( David et al . , 2005; Gabay et al . , 2011; Hampoelz et al . , 2005; Wang et al . , 2005 ) . Only the Drosophila Gαi protein forms a complex with Pins–Mud ( Schaefer et al . , 2001 ) , and Drosophila Gαi , but not Gαo , depends on RIC-8 for its cortical localization ( David et al . , 2005 ) . Seemingly unifying these independently described RIC-8 functions , C . elegans RIC-8 was shown to exhibit both GEF and chaperone activity , depending on the Gα subunit ( Afshar et al . , 2004; Afshar et al . , 2005 ) . The C . elegans GOA-1 and GPA-16 Gα proteins act in a substantially redundant manner in spindle positioning , but diverge in other ways . GPA-16 is closest to the Gαi class , has been implicated only in spindle positioning , and depends on RIC-8 for its cortical localization ( Afshar et al . , 2005; Bergmann et al . , 2003; Gotta and Ahringer , 2001 ) . Thus , both C . elegans and Drosophila RIC-8 appears to act as a chaperone for Gαi to promote spindle positioning . Also similar to Drosophila , the Gαo GOA-1 subunit does not require RIC-8 for its membrane localization ( David et al . , 2005; Afshar et al . , 2005 ) . Instead , RIC-8 was reported to act as a GEF for GOA-1 ( Afshar et al . , 2004 ) , and is usually considered to act as a GEF for Gαq and Gαo in neurotransmitter release . However , the contribution of a Gαo GEF in spindle positioning would mean that either Gα∙GTP or Gα∙GDP/GTP cycling promotes spindle pulling forces . Both of these possibilities seem unlikely in light of the results described here . The fact that Gα can be replaced with a PH–membrane anchor dismisses a general requirement for Gα∙GTP in pulling force generation . Moreover , RIC-8 functioning in a Gα∙GDP/GTP cycle is not supported by our knockout and membrane localization studies . Therefore , there is reason to question whether RIC-8 really functions as a GEF . The strongest support for such a function has come from in vitro experiments , in which RIC-8 showed rather inefficient GEF activity towards Gα∙GDP ( Kant et al . , 2016; Thomas et al . , 2008 ) . It appears conceivable that incubation of Gα with a chaperone that affects its folding causes nucleotide dissociation . If correct , the membrane localized GOA-1 in ric-8 mutants would reflect an incompletely functional form . Despite the observed replaceability of Gα with a general membrane anchor , Gα∙GTP has been reported to affect the spindle orientation in specific tissues ( Katanaev et al . , 2005; Schaefer et al . , 2001; Yoshiura et al . , 2012 ) . In Drosophila neuroblasts and sensory organ precursor cells , canonical G-protein signaling is used to align cellular polarity with tissue polarity ( Katanaev et al . , 2005; Yoshiura et al . , 2012 ) . As such , Gα∙GTP may indirectly contribute to spindle positioning . In addition , one of the Drosophila Pins GoLoco domains was found to preferentially interact with Gαo∙GTP , thereby linking the spindle positioning machinery with canonical G-protein signaling ( Kopein and Katanaev , 2009; Yoshiura et al . , 2012 ) . Thus , while not needed for the generation of dynein-dependent cortical pulling forces , further studies will need to reveal to what extent Gα∙GTP contributes to spindle positioning in a tissue or developmental context specific manner . For our in vivo dissection of spindle positioning , we developed and applied methods for germline-specific gene knockout , tagging of endogenous proteins , reliable expression of foreign sequences in the germline , and light-inducible protein heterodimerization . These methods further expand the molecular biology toolbox for in vivo studies and can be broadly applied to other biological processes . Of particular interest is the acquired possibility to experimentally control the position of the spindle , for instance for future studies aimed at deciphering how the spindle determines the plane of cell cleavage , and whether specific cell-cell contacts affect cell fate .
The names and associated genotypes of C . elegans strains used in this study are included in Supplementary file 1 . Animals were maintained at either 15 or 20˚C as described previously ( Brenner , 1974 ) . Strains expressing both ePDZ and LOV protein motifs were regarded as light-sensitive and thus cultured in the dark , and transferred while using red light only . Animals were kept on plates that contained nematode growth medium ( NGM ) that had been seeded with OP50 Escherichia coli bacteria . DNA vector-based repair templates to be used for CRISPR/Cas9-mediated genome editing were designed in A plasmid Editor ( M . Wayne Davis ) to include 500–1500 bp homology arms . These and all other sequences used were generated starting from either purified C . elegans genomic DNA or pre-existing vectors via PCR amplification using Q5 Hot Start High-Fidelity DNA Polymerase ( New England Biolabs ) . A list of all cloning , repair template and genotyping primers ( Integrated DNA technologies ) and DNA templates used has been included in Supplementary file 2 . PCR fragments were gel purified ( Qiagen ) , their concentrations measured using a BioPhotometer D30 ( Eppendorf ) and then ligated into pBSK by Gibson assembly ( New England Biolabs ) . gRNA vectors were generated by annealing of antisense oligonucleotide pairs and subsequent ligation into BbsI-linearized pJJR50 or BsaI-linearized pMB70 using T4 ligase ( New England Biolabs ) . All DNA vectors used for genome editing were transformed into DH5α competent cells and subsequently purified by midiprep ( Qiagen ) . Custom Perl scripts were written to design germline-optimized coding sequences according to the algorithm described in the legend of Figure 2—figure supplement 1 . After designing each coding sequence , we inserted either ( 1 ) normal synthetic introns with the sequence gtaagttt ( n36 ) ttttcag , where n36 is a 36 bp random DNA sequence with 30% GC content; or ( 2 ) PATC introns ( Frøkjær-Jensen et al . , 2016 ) . Our design algorithm is accessible via a web interface at http://104 . 131 . 81 . 59/ , and the source code can be found at https://github . com/dannyhmg/germline; copy archived athttps://github . com/elifesciences-publications/germline . Germline-optimized sequences were synthesized as gBlocks ( Integrated DNA Technologies ) and single-copy transgenes were generated using standard methods ( Frøkjær-Jensen et al . , 2012 ) . Please refer to Supplementary file 2 for detailed sequence features of each transgene . loxP and loxN sequences were integrated in the endogenous loci of essential genes ( see CRISPR/Cas9-mediated genome editing section for details ) . For the FLP recombinase , the hyperactive FLP G5D variant ( Schwartz and Jorgensen , 2016 ) was used ( pMLS262; Addgene #73718 ) . For germline-specific expression , we used a long version of the pie-1 promoter including enhancer sequences ( pAZ132 , a kind gift from A . A . Hyman ) . To initiate the recombination cascade , the germline-specific FLP expression vector was injected in P0 mothers with the following protocol to favor germline expression ( Personal communication Oliver Hobert , Worm Breeders Gazette , February 21 , 2013 ) ; linearized FLP construct ( 2 ng/µl ) , PvuII digested E . coli genomic DNA ( 150 ng/µl ) , co-injection marker Pmyo-2::tdtomato ( 2 ng/µl ) . Transgenic F1 animals were singled and allowed to lay eggs for at least 24 hr . From F1 with 100% embryonic lethal broods ( ric-8 and rgs-7 are essential for embryogenesis ) , early embryos were isolated and used for spindle severing experiments . Either the wild type N2 or SV1818 ( pha-1 ( e2123ts ) 4x outcrossed ) C . elegans genetic background was used for the generation of CRISPR/Cas9 alleles . Injection mixes with a total volume of 50 µl were prepared in MilliQ HR2RO and contained a combination of 50 ng/µl Peft-3:cas9 ( Addgene ID #46168 ( Friedland et al . , 2013 ) or 60 ng/µl pJW1285 ( Addgene ID #61252 [Dickinson et al . , 2013] ) , 50–100 ng/µl u6::sgRNA ( targeting genomic sequences listed in Supplementary file 2 ) , 50 ng/µl of ( PAGE-purified oligonucleotide ) repair template and 2 . 5 ng/µl of the co-injection pharyngeal marker myo-2p::tdtomato . Injection mixes were spun down in a microcentrifuge ( Eppendorf ) for at least 10 min at 13 , 000 RPM prior to use . Young adult hermaphrodites were injected in the germline using an inverted micro-injection setup ( Eppendorf ) . After injection , animals were singled and grown at 15 or 20˚C . F1 animals were then picked to a total of at least 96 , and grown with two or three animals per plate for 7–8 days at 20˚C until freshly starved . Half a plate containing F2 and F3 animals was then washed off with M9 medium supplemented with 0 . 05% Tween-20 , and subsequently lysed to extract genomic DNA . Some knock-ins were obtained using co-CRISPR selection: rescue of pha-1 ( e2123ts ) ( Ward , 2015 ) , generation of visible unc-22 ( Kim et al . , 2014 ) or dpy-10 ( Paix et al . , 2015 ) phenotypes , or integration of an self-excisable cassette carrying a visible marker ( Dickinson et al . , 2015 ) . Genotyping was carried out by PCR amplification with OneTaq polymerase ( New England Biolabs ) of genome sequences using primers annealing in the inserted sequence and a genomic region not included in the repair template . Confirmed alleles were subsequently sequenced ( Macrogen Europe ) . Prior to live imaging , embryos were dissected from adult hermaphrodites onto coverslips ( Menzel-Gläser ) in 0 . 8x egg salts buffer ( 94 mM NaCl , 32 mM KCl , 2 . 7 mM CaCl2 , 2 . 7 mM MgCl2 , 4 mM HEPES , pH 7 . 5; ( Tagawa et al . , 2001 ) or M9 , and mounted on 4% agarose pads . Spinning disk imaging of embryos was performed using a Nikon Eclipse Ti with Perfect Focus System , Yokogawa CSU-X1-A1 spinning disk confocal head , Plan Apo VC 60x N . A . 1 . 40 oil and S Fluor 100x N . A . 0 . 5–1 . 3 ( at 1 . 3 , used for UV-laser photo-ablation ) objectives , Photometrics Evolve 512 EMCCD camera , DV2 two-channel beam-splitter for simultaneous dual-color imaging , Vortran Stradus 405 nm ( 100 mW ) , Cobolt Calypso 491 nm ( 100 mW ) , Cobolt Jive 561 nm ( 100 mW ) , Vortran Stradus 642 nm ( 110 mW ) and Teem Photonics 355 nm Q-switched pulsed lasers controlled with the ILas system ( Roper Scientific France/PICT IBiSA , Institut Curie , used for photo-ablation ) , ET-GFP ( 49002 ) , ET-mCherry ( 49008 ) , ET-GFPmCherry ( 49022 ) and ET-Cy5 ( 49006 ) filters , ASI motorized stage MS-2000-XYZ with Piezo Top Plate , and Sutter LB10-3 filter wheel . The microscope was operated using MetaMorph 7 . 7 software and situated in a temperature-controlled room ( 20˚C ) . The temperature of the stage and objective was controlled at 25˚C with a Tokai Hit INUBG2E-ZILCS Stage Top Incubator during experiments . Images were acquired in either streaming mode with 250 or 500 ms exposure , or time-lapse mode with 250 , 500 or 1500 ms exposure and 2 or 5 s intervals . Laser power and exposure times were kept constant within experiments . For the quantification of membrane invaginations embryos were imaged by 250 ms exposure stream acquisition starting in the DNA plane at anaphase onset , as judged by GFP::tubulin signal . During anaphase , the spinning disk imaging plane was moved as close to the membrane as possible while keeping the cytosol discernable from the membrane signal . Acquisitions were terminated at early telophase , as judged by the PH::eGFP::LOV signal . For experiments involving balanced epdz::mcherry::dhc-1/+ , each animal was confirmed to be positive for epdz::mcherry::dhc-1 by fluorescence before the experiment . Images acquired by spinning disk microscopy were rotated , cropped , annotated , provided with scale bars , and processed further by linear adjustment of brightness and contrast using ImageJ and FIJI . Fluorophores used in this study include ( e ) GFP , mCherry , Alexa-488 , Alexa-568 and Atto 647N . For immunohistochemistry experiments L4 hermaphrodites were grown on RNAi plates seeded with HT115 Escherichia coli bacteria strains generating double-stranded RNA ( dsRNA ) targeting genes of interest ( goa-1 , gpa-16 , gpr-1 ) for 48 hr at 15˚C prior to fixation ( Timmons and Fire , 1998 ) . For all other gene knock-down experiments , young adult hermaphrodites were injected with dsRNA targeting genes of interest ( goa-1 , gpa-16 , gpr-1 , ric-8 , rgs-7 ) and grown for 48 hr at 15˚C ( Fire et al . , 1998 ) prior to experiments . To generate dsRNA , coding regions of genes of interest were PCR amplified using Q5 Hot Start High-Fidelity DNA Polymerase ( New England Biolabs ) . These PCR products were used as templates for in vitro dsRNA synthesis ( MEGAscript T7 transcription kit , ThermoFisher Scientific ) . dsRNA was diluted 5x in DEPC HR2RO prior to micro-injection . ORF clones from the Vidal and Ahringer RNAi libraries were used ( Kamath et al . , 2003; Rual et al . , 2004 ) . Mitotic spindle severing was performed in essence as described ( Grill et al . , 2001; Portegijs et al . , 2016 ) . One-cell embryos expressing GFP- or mCherry-labeled tubulin were imaged during mitosis using the spinning disk microscope setup described above , equipped with a Teem Photonics 355 nm Q-switched pulsed laser controlled with the ILas system ( Roper Scientific France/PICT IBiSA , Institut Curie ) . At anaphase onset , as judged by spindle morphology and mobility , spindles were severed as shown in Figure 1c and Video 2 . Centrosome displacement was recorded by 500 ms exposure streaming acquisition , and peak velocities were subsequently extrapolated using the FIJI TrackMate plugin . Dark state experiments were performed on the spinning disk setup described above . For local photoactivation of LOV2 in C . elegans embryos , light was applied in a region of variable size depending on each individual experiment using a 491 nm laser controlled with the ILas system ( Roper Scientific France/PICT IBiSA , Institut Curie ) . Due to high sensitivity of LOV2 to blue light and variations in laser power , embryos of strain SV2061 ( expressing diffuse ePDZ::mCherry and PH::eGFP::LOV ) were used to calibrate the amount of laser power required for local activation of LOV2 prior to experiments . During both global and local photoactivation assays and dark state spindle severing experiments embryos were kept away from blue light as much as practically feasible . To this end , aluminum foil was used to cover the microscope setup , and optical filters were inserted in the light path to remove LOV2-activating wavelengths from the transmitted light used to locate embryos on slides . Prior to experimental use of embryos , unintended premature cortical recruitment of ePDZ-mCherry or ePDZ-mCherry-LIN-5 was assessed by observation of mCherry localization patterns . For immunostaining of C . elegans embryos , embryos were dissected from adults in 10 µl MilliQ HR2RO on slides coated with poly-L-lysine . Samples were then freeze-cracked and fixed in methanol for 5 min . at −20˚C and subsequently in acetone for 5 min . at −20˚C . Embryos were then rehydrated in phosphate buffered saline +0 . 05% Tween-20 ( PBST ) , blocked for 1 hr at 4˚C in PBST +1% bovine serum albumin and 1% goat serum ( Sigma-Aldrich ) , and then incubated at room temperature with primary antibodies for 1 hr and then with secondary antibodies for 45 min . , both in blocking solution , with four 10 min washes in PBST following each antibody mix . Finally , embryos were embedded in ProLong Gold Antifade with DAPI . Primary antibodies used were mouse anti-LIN-5 [1:10 , ( Lorson et al . , 2000 ) , mouse anti-FLAG M2 ( Sigma-Aldrich ) and rabbit anti-DHC-1 ( 1:100 ( Gonczy et al . , 1999 ) ; a kind gift from P . Gönczy ) . Secondary antibodies used were goat anti-rabbit Alexa-488 , goat anti-rabbit Alexa-568 , goat anti-mouse Alexa-488 , goat anti-mouse Alexa-568 ( Invitrogen ) and goat anti-mouse Atto 647N ( Sigma-Aldrich ) , all at 1:500 dilution in blocking solution . Imaging of immunolabeled embryos was performed on the spinning disk setup described above . All quantitative spinning disk image analyses were performed in either ImageJ or FIJI . For quantification of membrane invaginations , movies were limited to the 200 frames ( 50 s ) preceding the onset of telophase . Images were then cropped to include the outer limits of the PH::eGFP signal . Transient cortical dots were tracked manually using the MTrackJ ImageJ plugin . Larger , more static structures result from membrane ruffles , which are distinct from the more dynamic invaginations , as can be seen in Video 11 . To yield the distribution of invaginations on the length axis of the visible embryo cortex , recorded x coordinates were incremented into groups of 5% embryo length each . To quantify the cortical recruitment and dynamics of ePDZ::mCherry , ePDZ::mCherry::GPR-1 , LIN-5::ePDZ::mCherry and ePDZ::mCherry::DHC-1 by PH::eGFP::LOV , multiple 20 px wide linescans were drawn perpendicular to the membrane per analyzed embryo . An intensity profile was plotted per linescan at each acquired time point , from each of which an average of the maximum three pixel values was extracted to yield the peak intensity values at the membrane . Each intensity measurement was first corrected for background noise with a value measured outside of the embryo in a 50 × 50 px region of interest , and cortex to cytoplasm intensity ratios were calculated using average cytoplasmic intensity measurements in a 50 × 50 or 29 × 23 px region of interest at all timepoints analyzed . Fluorescence intensity measurements as measure for Cre ( FLPon ) activation ( Figure 1—figure supplement 2 ) were taken as total embryo average intensity minus background signal using ImageJ measurement tool . The half time of ePDZ-LOV interaction after a pulse activation was inferred from a non-linear , single component regression . All numerical data processing and graph generation was performed using Excel 2011 ( Microsoft ) and Prism 7 ( GraphPad software , inc . ) . All data were shown as means with SEM . Statistical significance as determined using two-tailed unpaired Student’s t-tests , Mann-Whitney U tests and the Wilcoxon matched-pairs signed rank test . Correlation coefficients between two data sets were calculated using Pearson r correlation tests or Spearman rank correlation tests . Data sets were assessed for their fit to a Gaussian distribution using the D’Agostino-Pearson omnibus K2 normality test prior to application of appropriate statistical test . A p-value of <0 . 05 was considered significant . *p<0 . 05; **p<0 . 01; ***p<0 . 001; ****p<0 . 0001 . All statistical analyses were performed in Prism 7 ( GraphPad software , inc . ) . Our design algorithm is accessible via a web interface at http://104 . 131 . 81 . 59/ , and the source code can be found at https://github . com/dannyhmg/germline; copy archived at https://github . com/elifesciences-publications/germline . The data that support the findings of this study are included in the supplementary information .
The position of the mitotic spindle determines the plane of cell cleavage , and thereby daughter cell location , size , and content . Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules , which requires an evolutionarily conserved complex of Gα∙GDP , GPR-1/2Pins/LGN , and LIN-5Mud/NuMA proteins . To examine individual functions of the complex components , we developed a genetic strategy for light-controlled localization of endogenous proteins in C . elegans embryos . By replacing Gα and GPR-1/2 with a light-inducible membrane anchor , we demonstrate that Gα∙GDP , Gα∙GTP , and GPR-1/2 are not required for pulling-force generation . In the absence of Gα and GPR-1/2 , cortical recruitment of LIN-5 , but not dynein itself , induced high pulling forces . The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane . Our results define Gα∙GDP–GPR-1/2Pins/LGN as a regulatable membrane anchor , and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces .
A cell about to divide must decide where exactly to cut itself in two . Split right down the middle , and the two daughter cells will be identical; offset the cleavage plane to one side , and the resulting siblings will have different sizes , places and fates . In animals , the splitting of cells is dictated by the location of the spindle , a structure that forms when cable-like microtubules stretch from the cell membrane to attach to the chromosomes . At the membrane , a group of proteins tugs on the microtubules to bring the spindle into the correct position . One of these proteins , dynein , is a motor that uses microtubules as its track to pull the spindle into place . What the other parts of the complex do is still unclear , but a general assumption is that they may be serving as an anchor for dynein . To test this model , Fielmich , Schmidt et al . removed one or more proteins from the complex in the developing embryos of the nematode worm Caenorhabditis elegans . A light-activated system then linked the remaining proteins to the membrane by tying them to an artificial anchor . Two of the proteins in the complex could be replaced with the artificial anchor , but pulling forces were absent when dynein was artificially tied to the membrane . This indicates that the motor being anchored at the edge of the cell is not enough for it to pull on microtubules . Instead , the experiments showed that dynein needs to be activated by another component of the complex , a protein called LIN-5 . This suggests that individual proteins in the complex have specialized roles that go beyond simply tethering dynein . In fact , steering where LIN-5 was attached on the membrane helped to control the location of the spindle , and therefore of the cleavage plane . As mammals have a protein similar to LIN-5 , dissecting the roles of the components involved in positioning the spindle in C . elegans could help to understand normal and abnormal human development . In addition , these results demonstrate that creating artificial interactions between proteins using light is a powerful technique to study biological processes .