Molecular Defects in the Motor Adaptor BICD2 Cause Proximal Spinal Muscular Atrophy with Autosomal-Dominant Inheritance
2013; Elsevier BV; Volume: 92; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2013.04.013
ISSN1537-6605
AutoresKoen Peeters, Ivan Litvinenko, Bob Asselbergh, Leonardo Almeida-Souza, Teodora Chamova, Thomas Geuens, Elke Ydens, M. Zimoń, Joy Irobi, Els De Vriendt, Vicky De Winter, Tinne Ooms, Vincent Timmerman, Ivailo Tournev, Albena Jordanova,
Tópico(s)Congenital heart defects research
ResumoThe most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals’ lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans. The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals’ lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans. Inherited spinal muscular atrophies (SMAs) form a diverse group of disorders characterized by muscle weakness and atrophy caused by the degeneration of anterior horn cells.1Dubowitz V. Ramblings in the history of spinal muscular atrophy.Neuromuscul. Disord. 2009; 19: 69-73Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 2Kolb S.J. Kissel J.T. Spinal muscular atrophy: a timely review.Arch. Neurol. 2011; 68: 979-984Crossref PubMed Scopus (199) Google Scholar Although the most common form of SMA is an autosomal-recessive condition associated with loss-of-function mutations in SMN1 (MIM 600354) in chromosomal region 5q13,3Viollet L. Barois A. Rebeiz J.G. Rifai Z. Burlet P. Zarhrate M. Vial E. Dessainte M. Estournet B. Kleinknecht B. et al.Mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.Ann. Neurol. 2002; 51: 585-592Crossref PubMed Scopus (44) Google Scholar rare families with dominant inheritance have been reported.4Takashima H. 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J. Hum. Genet. 2007; 81: 67-76Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar whereas the proximal forms are caused by mutations in VAPB (MIM 605704),5Nishimura A.L. Mitne-Neto M. Silva H.C. Richieri-Costa A. Middleton S. Cascio D. Kok F. Oliveira J.R. Gillingwater T. Webb J. et al.A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis.Am. J. Hum. Genet. 2004; 75: 822-831Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar LMNA (MIM 150330),16Rudnik-Schöneborn S. Botzenhart E. Eggermann T. Senderek J. Schoser B.G. Schröder R. Wehnert M. Wirth B. Zerres K. Mutations of the LMNA gene can mimic autosomal dominant proximal spinal muscular atrophy.Neurogenetics. 2007; 8: 137-142Crossref PubMed Scopus (28) Google Scholar and TRPV4 (MIM 605427)6Deng H.X. Chen W. Hong S.T. Boycott K.M. Gorrie G.H. Siddique N. Yang Y. Fecto F. Shi Y. Zhai H. et al.Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.Nature. 2011; 477: 211-215Crossref PubMed Scopus (871) Google Scholar or are linked to unresolved loci.4Takashima H. Nakagawa M. Suehara M. Saito M. Saito A. Kanzato N. Matsuzaki T. Hirata K. Terwilliger J.D. Osame M. Gene for hereditary motor and sensory neuropathy (proximal dominant form) mapped to 3q13.1.Neuromuscul. Disord. 1999; 9: 368-371Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7Harms M.B. Allred P. Gardner Jr., R.J. Fernandes Filho J.A. Florence J. Pestronk A. Al-Lozi M. Baloh R.H. Dominant spinal muscular atrophy with lower extremity predominance: linkage to 14q32.Neurology. 2010; 75: 539-546Crossref PubMed Scopus (48) Google Scholar, 17Penttilä S. Jokela M. Hackman P. Maija Saukkonen A. Toivanen J. Udd B. Autosomal dominant late-onset spinal motor neuronopathy is linked to a new locus on chromosome 22q11.2-q13.2.Eur. J. Hum. Genet. 2012; 20: 1193-1196Crossref PubMed Scopus (11) Google Scholar Furthermore, several clinical entities remain without a molecular genetic diagnosis.8Darras B.T. Non-5q spinal muscular atrophies: the alphanumeric soup thickens.Neurology. 2011; 77: 312-314Crossref PubMed Scopus (34) Google Scholar We studied a four-generation Bulgarian family (with Turkish ethnic background) afflicted with autosomal-dominant proximal SMA (Figure 1A). Disease status in the affected individuals was determined by clinical examination and electrophysiological evaluation. All clinical findings are summarized in Table 1.Table 1Clinical and Electrophysiological Features of the Individuals with BICD2 MutationsAffected Individual177.II.4177.III.1177.III.4177.III.5177.III.6177.III.8177.IV.3177.IV.4177.IV.5177.IV.6177.IV.7584.II.3Age of onset (years)225644115242Age at assessment (years)4844302424296563542Waddling gait++++++++++++CPK levelND1,110178NDNDND12051123NDNDNDFasciculations−+−−−−−−−−−+Muscle Weakness (MRC Scale)Proximal muscles in LLs444444444+43Distal muscles in LLs545555555−54Axial muscles445555555−53ReflexesPatellar−+/−+/−++/−+/−+/−+/−+/−ND+/−−Achilles−−−+/−+/−+/−+/−+/−+/−ND+/−−Deformities and PostureScapular winging++−+++++++++Lumbal hyperlordosis−+−++−++++++HypotrophiesProximal muscles in LLs++−++++−+−−+Distal muscles in LLs−+−++−+−−−−+Nerve Conduction Studies and EMGMCV of the nervous peroneusND43.5NDNDNDND54.858.355.1NDND45.0Anterior horn involvementND++NDNDND+++NDND+Normal CPK range: 20–200. The following abbreviations are used: −, absent; +, present; +/−, moderately reduced; MRC, Medical Research Council; LL, lower limb; CPK, creatine phosphokinase; EMG, electromyography; MCV, motor conduction velocity; and ND, not determined. Open table in a new tab Normal CPK range: 20–200. The following abbreviations are used: −, absent; +, present; +/−, moderately reduced; MRC, Medical Research Council; LL, lower limb; CPK, creatine phosphokinase; EMG, electromyography; MCV, motor conduction velocity; and ND, not determined. The onset age varied between 1 and 6 years (mean 3.17 ± 1.70 years), and individuals presented with delayed motor milestones, such as walking (in persons 177.III.1, 177.IV.3, and 177.IV.4), difficulties in getting upright from a squatting position (Gowers’ sign) and in climbing stairs, a waddling gait, and slow running. The leg weakness did not progress significantly over time, given that affected individuals still remained ambulatory even into the fifth decade. Upon neurologic examination, muscle weakness was limited to the lower extremities and showed predominant involvement in the proximal leg muscles (Figures 1B and 1C). Distal muscle weakness in the legs (score 4/5 on the Medical Research Council [MRC] scale for muscle strength) was found in one of the oldest family members (177.III.1). Axial muscle involvement, with difficulties in getting up from a lying position, was present upon disease progression. Bulbar muscles were spared in all affected individuals. Other muscles, including those of the face and upper extremities, showed normal strength. Patellar tendon reflexes were depressed in nine persons, whereas Achilles hyporeflexia or areflexia was found in all of them. Reflexes in the upper limbs were preserved. Fasciculations were observed in the proximal muscles of the upper limbs in person 177.III.1. Skeletal deformities (lumbal hyperlordosis and scapular winging) were present in most affected individuals. Symmetric wasting was most prominent in the hip muscles, whereas distal leg muscles seemed atrophic in the older family members. Sensation was normal for all modalities. Serum creatine phosphokinase (CPK) levels were mostly normal. We performed electromyography (EMG) studies while maintaining skin temperature over the first dorsal interosseous muscle at >30.5°C on a Toennies NeuroScreen System (Jaeger GnbH). Sensory and motor nerve conduction studies showed normal conduction velocities and amplitudes for the upper and lower extremities (Figures S1A and S1B, available online). EMG showed predominantly large-amplitude and long-duration motor unit potentials in the muscles of the lower and upper extremities, in keeping with anterior horn cell damage (Figures S1C and S1D). All study participants or their legal representatives provided written informed consent prior to enrollment. Local institutional review boards approved this study. Genomic DNA was isolated from peripheral blood according to standard procedures. We performed genome-wide multipoint parametric linkage analysis on family 177 with Allegro v.1.2c in the easyLinkage software18Lindner T.H. Hoffmann K. easyLINKAGE: a PERL script for easy and automated two-/multi-point linkage analyses.Bioinformatics. 2005; 21: 405-407Crossref PubMed Scopus (151) Google Scholar by using an in-house-developed panel of 436 polymorphic short-tandem-repeat (STR) markers under an autosomal-dominant model, equal male and female recombination rates and allele frequencies, and a 0.0001 disease frequency. Because the disease was presumed to have originated de novo in the proband, the disease status of her unaffected siblings and parents was put as “unknown.” The initial analysis showed only one suggestive linkage peak with a LOD ≥ 2.00 on chromosome 9q (LOD = 2.71) (Figure S2A). After fine mapping with additional STR markers, the disease-segregating locus could be delineated to a 12.7 Mb region containing 132 distinct genes between D9S1812 and D9S176 (Figure 1A and Figure S2B). The TargetSeq Exome Enrichment System was used for library preparation, capture, and enrichment of about 45.1 Mb of coding regions from two affected brothers (177.IV.5 and 177.IV.6). Fragment reads (75 + 35 bp) were sequenced on a SOLiD 5500xl instrument. Mapping to the human reference genome (GRCh37) and variant calling were performed with the CLC Genomics Workbench. Subsequent annotation and filtering were executed with GenomeComb.19Reumers J. De Rijk P. Zhao H. Liekens A. Smeets D. Cleary J. Van Loo P. Van Den Bossche M. Catthoor K. Sabbe B. et al.Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing.Nat. Biotechnol. 2012; 30: 61-68Crossref Scopus (167) Google Scholar We extracted heterozygous, novel variants (defined as a frequency ≤ 1% in public databases such as dbSNP137 and 1000 Genomes Project and our in-house collection of genomes) that were shared between both affected siblings, were located within the linkage interval, and had an impact on the protein level (nonsynonymous variations and splice-site changes). The analysis revealed that the linkage region contained 50 shared variations, of which only one nonsynonymous variant was novel: c.320C>T (p.Ser107Leu) in bicaudal D homolog 2 (Drosophila) (BICD2 [MIM 609797; RefSeq accession number NM_015250.3]). This missense mutation cosegregated with disease in the family, as shown by bidirectional sequencing with the BigDye Terminator v.3.1 Cycle Sequencing kit on an ABI3730xl DNA Analyzer (Applied Biosystems), and was, as presumed, a de novo event in the disease founder, 177.II.4 (Figures 1A and 1E). It was absent in a cohort of 289 ancestry-matched controls (51 Bulgarian Turks, 51 Bulgarians, and 187 Turks). Interestingly, the c.320C>T BICD2 mutation is positioned within a CpG dinucleotide. Given that an estimated 80% of CpG cytosines are methylated in human cells,20Ehrlich M. Gama-Sosa M.A. Huang L.H. Midgett R.M. Kuo K.C. McCune R.A. Gehrke C. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.Nucleic Acids Res. 1982; 10: 2709-2721Crossref PubMed Scopus (764) Google Scholar the c.320 position could as such be a potential mutational “hotspot,” prone to C>T transitions through spontaneous deamination of 5-methylcytosine.21Cooper D.N. Youssoufian H. The CpG dinucleotide and human genetic disease.Hum. Genet. 1988; 78: 151-155Crossref PubMed Scopus (794) Google Scholar, 22Coulondre C. Miller J.H. Farabaugh P.J. Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli.Nature. 1978; 274: 775-780Crossref PubMed Scopus (873) Google Scholar In order to obtain further genetic evidence of BICD2-dependent pathogenicity, we performed an unbiased screen of all coding exons and exon-intron boundaries of BICD2 (primers are available upon request) in a cohort of 119 unrelated Bulgarian simplex cases who presented with clinical and EMG features of progressive anterior horn involvement and who had been excluded for SMN1 deletions. In 113 (95%) individuals, the clinical picture was dominated by proximal muscle weakness, whereas in 6 (5%) persons, distal muscles were mostly affected. The subjects with proximal SMA were further classified as having type 1 (27.4%), type 2 (14.1%), type 3 (55.8%), or type 4 (2.7%). In this cohort, we identified a second heterozygous missense mutation in BICD2, c.2321A>G (p.Glu774Gly), in a Bulgarian simplex case diagnosed with proximal SMA (Figures 1D and 1E). Haplotype analysis suggested that this mutation also originated de novo in the proband (584.II.3) (Figure 1D). The variant was absent in 304 control individuals (108 Bulgarians, 49 Bulgarian Turks, and 147 Turks). In silico prediction of the functional effect of both mutations with PolyPhen-223Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9290) Google Scholar and MutationTaster24Schwarz J.M. Rödelsperger C. Schuelke M. Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations.Nat. Methods. 2010; 7: 575-576Crossref PubMed Scopus (2123) Google Scholar showed that they target highly conserved nucleotide and amino acid residues and are therefore very likely to be deleterious. The clinical course of person 584.II.3, who harbors the BICD2 c.2321A>G (p.Glu774Gly) mutation, was extremely similar to that of individuals with the c.320C>T (p.Ser107Leu) mutation. This person was initially diagnosed with the Kugelberg-Welander type of SMA (MIM 253400). She presented with delayed motor milestones, a waddling gait, Gowers’ sign, and proximal upper-limb fasciculations. Neurological examination showed predominantly proximal muscle weakness (3/5 on the MRC scale) and some distal muscle involvement, comparable to that of an age-matched affected individual in family 177 (177.III.1). EMG analysis showed anterior horn involvement. BICD2, an evolutionary conserved motor-adaptor protein, is implicated in dynein-mediated transport as a molecular linker between the dynein motor and various cargos.25Hoogenraad C.C. Akhmanova A. Howell S.A. Dortland B.R. De Zeeuw C.I. Willemsen R. Visser P. Grosveld F. Galjart N. Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.EMBO J. 2001; 20: 4041-4054Crossref PubMed Scopus (236) Google Scholar, 26Matanis T. Akhmanova A. Wulf P. Del Nery E. Weide T. Stepanova T. Galjart N. Grosveld F. Goud B. De Zeeuw C.I. et al.Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex.Nat. Cell Biol. 2002; 4: 986-992Crossref PubMed Scopus (310) Google Scholar, 27Hoogenraad C.C. Wulf P. Schiefermeier N. Stepanova T. Galjart N. Small J.V. Grosveld F. de Zeeuw C.I. Akhmanova A. Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport.EMBO J. 2003; 22: 6004-6015Crossref PubMed Scopus (160) Google Scholar, 28Splinter D. Tanenbaum M.E. Lindqvist A. Jaarsma D. Flotho A. Yu K.L. Grigoriev I. Engelsma D. Haasdijk E.D. Keijzer N. et al.Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry.PLoS Biol. 2010; 8: e1000350Crossref PubMed Scopus (211) Google Scholar According to tissue expression databases (GeneCards), mammalian BICD2 is widely present. We confirmed this finding via immunoblot analysis of BICD2 levels in a range of mouse tissues (Figure S3). In addition, we could demonstrate a high presence of BICD2 in the spinal cord, the afflicted tissue in SMA. BICD2 consists of three highly conserved coiled-coil regions, of which the N-terminal domain binds to the dynein motor,25Hoogenraad C.C. Akhmanova A. Howell S.A. Dortland B.R. De Zeeuw C.I. Willemsen R. Visser P. Grosveld F. Galjart N. Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.EMBO J. 2001; 20: 4041-4054Crossref PubMed Scopus (236) Google Scholar whereas the C-terminal coiled-coil domain interacts with various cargos, such as RAB6A (Figure 2A).26Matanis T. Akhmanova A. Wulf P. Del Nery E. Weide T. Stepanova T. Galjart N. Grosveld F. Goud B. De Zeeuw C.I. et al.Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex.Nat. Cell Biol. 2002; 4: 986-992Crossref PubMed Scopus (310) Google Scholar The BICD2 N terminus on its own has very strong dynein-recruiting activity and can chronically impair dynein-dynactin function, thus disturbing retrograde trafficking and causing cellular abnormalities such as Golgi fragmentation and neurofilament swellings.25Hoogenraad C.C. Akhmanova A. Howell S.A. Dortland B.R. De Zeeuw C.I. Willemsen R. Visser P. Grosveld F. Galjart N. Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.EMBO J. 2001; 20: 4041-4054Crossref PubMed Scopus (236) Google Scholar, 27Hoogenraad C.C. Wulf P. Schiefermeier N. Stepanova T. Galjart N. Small J.V. Grosveld F. de Zeeuw C.I. Akhmanova A. Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport.EMBO J. 2003; 22: 6004-6015Crossref PubMed Scopus (160) Google Scholar, 29Teuling E. van Dis V. Wulf P.S. Haasdijk E.D. Akhmanova A. Hoogenraad C.C. Jaarsma D. A novel mouse model with impaired dynein/dynactin function develops amyotrophic lateral sclerosis (ALS)-like features in motor neurons and improves lifespan in SOD1-ALS mice.Hum. Mol. Genet. 2008; 17: 2849-2862Crossref PubMed Scopus (69) Google Scholar Full-length BICD2 does not display such perturbing activities, presumably because the C terminus regulates motor-complex binding and inhibits the N terminus in the native state until after cargo loading.25Hoogenraad C.C. Akhmanova A. Howell S.A. Dortland B.R. De Zeeuw C.I. Willemsen R. Visser P. Grosveld F. Galjart N. Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.EMBO J. 2001; 20: 4041-4054Crossref PubMed Scopus (236) Google Scholar Interestingly, the targeted residues are positioned in two different interaction domains of BICD2. Whereas the p.Ser107Leu substitution lies within the N-terminal dynein-binding region, the p.Glu774Gly alteration is located in the C-terminal cargo-loading domain (Figure 2A). To study the functional effect of these amino acid substitutions on BICD2 binding properties, we performed coimmunoprecipitation (coIP) and colocalization studies in cellular models. To this end, BICD2 expression constructs in pLenti6/V5-DEST or pLenti6/EGFP-DEST were generated by Gateway cloning with full-length human BICD2 cDNA from the IMAGE Human cDNA clone 6169195 (Thermo Fischer Scientific). Mutations were introduced into the human transcript by site-directed mutagenesis, and stable SH-SY5Y cell lines were created by lentiviral transduction as described.30Salmon P. Trono D. Production and titration of lentiviral vectors.Curr. Protoc. Neurosci. 2006; 37: 4.21.1-4.21.24Google Scholar For the immunoprecipitation experiments, human neuroblastoma (SH-SY5Y) cells stably expressing wild-type or altered BICD2 proteins were grown in modified Eagle’s medium (Invitrogen) under blasticidin selection. Cell lysates were incubated with Anti-V5 Agarose (Sigma-Aldrich) or Protein G Sepharose 4 Fast Flow beads (GE Healthcare) with monoclonal mouse dynein intermediate chain 1 (DIC) antibody (Abcam) or polyclonal rabbit RAB6 antibody (Cell Signaling) according to standard procedures. We detected enhanced coprecipitation of the p.Ser107Leu altered protein, compared to wild-type BICD2, by endogenous DIC pulldown. In a reciprocal experiment pulling down BICD2-V5, higher amounts of DIC coprecipitated with p.Ser107Leu BICD2 than with the wild-type protein (Figure 2B). To explore whether this altered N-terminal BICD2 interaction is relevant for the pathophysiology in humans, we further analyzed lymphoblast cultures derived from affected individuals with the same p.Ser107Leu substitution. Consistent with our findings in the BICD2 cellular model, coIP of BICD2 with DIC showed their enhanced interaction in protein extracts of three affected persons compared to a healthy family member (Figure 2C). The p.Glu774Gly alteration, on the other hand, is localized in the BICD2 C-terminal domain, known to directly interact with RAB6A, a regulator of Golgi-ER trafficking.26Matanis T. Akhmanova A. Wulf P. Del Nery E. Weide T. Stepanova T. Galjart N. Grosveld F. Goud B. De Zeeuw C.I. et al.Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex.Nat. Cell Biol. 2002; 4: 986-992Crossref PubMed Scopus (310) Google Scholar Pulldown of endogenous RAB6A protein coimmunoprecipitated less p.Glu774Gly protein than wild-type BICD2 (Figure 2D). The interaction efficiency between p.Ser107Leu altered BICD2 and RAB6A was comparable to that between the wild-type protein and RAB6A (Figure 2D). To investigate whether these alterations in binding capacity affect the subcellular localization of BICD2, we performed immunocytochemistry experiments in neuronal (SH-SY5Y) and nonneuronal (HeLa) cell lines. HeLa cells were first transiently transfected with EGFP-tagged wild-type or altered BICD2 constructs with the use of Lipofectamine LTX reagent (Life Technologies). Immunostaining was performed with various antibodies (see below), and cells were imaged with a LSM700 confocal microscope (Zeiss) with identical acquisition parameters. In wild-type-expressing HeLa cells, BICD2-EGFP signal largely overlapped with the RAB6A signal (polyclonal rabbit RAB6A antibody, Cell Signaling), as reported before.26Matanis T. Akhmanova A. Wulf P. Del Nery E. Weide T. Stepanova T. Galjart N. Grosveld F. Goud B. De Zeeuw C.I. et al.Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex.Nat. Cell Biol. 2002; 4: 986-992Crossref PubMed Scopus (310) Google Scholar In line with its reduced coIP, the p.Glu774Gly altered BICD2, compared to the wild-type protein, showed a decreased colocalization with RAB6A, as illustrated by line intensity plots and quantification of fluorescence correlation (Figure 3). Intriguingly, the p.Ser107Leu altered BICD2 also showed significantly reduced colocalization with RAB6A-positive regions, albeit less pronounced than the other altered protein. In addition, only cells expressing p.Ser107Leu showed an intense punctual signal in the perinuclear region (Figure 3, see arrow). Further analysis demonstrated that this signal colocalized with the microtubule-organizing center (MTOC, as visualized with polyclonal rabbit γ-tubulin, Abcam), to which dynein motor transport is directed (Figure 4). Interestingly, previous studies with a truncated BICD
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