Structural Organization of the Actin-Spectrin–Based Membrane Skeleton in Dendrites and Soma of Neurons

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

Boran Hana,b,c,1, Ruobo Zhoua,b,c,1,2, Chenglong Xiaa,b,c, and Xiaowei Zhuanga,b,c,2

aHoward Hughes Medical Institute, Harvard University, Cambridge, MA 02138; bDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and cDepartment of Physics, Harvard University, Cambridge, MA 02138

Contributed by Xiaowei Zhuang, June 22, 2017 (sent for review March 27, 2017; reviewed by Bianxiao Cui and Velia M. Fowler) Actin, spectrin, and associated molecules form a membrane- flexible mechanical support important for the integrity of the associated periodic skeleton (MPS) in neurons. In the MPS, short axons under mechanical stress (1), which can explain why spectrin actin filaments, capped by actin-capping proteins, form ring-like deletion and mutations result in axon breakage and buckling during structures that wrap around the circumference of neurites, and these animal locomotion (17–19). The MPS also plays a role in shaping rings are periodically spaced along the neurite by spectrin tetramers, the axon morphology, and adducin depletion causes axon en- forming a quasi-1D lattice structure. This 1D MPS structure was largement (20). Notably, the MPS is able to organize other asso- initially observed in axons and exists extensively in axons, spanning ciated molecules, such as ankyrin, ion channels, and adhesion nearly the entire axonal shaft of mature neurons. Such 1D MPS was molecules, into a periodic distribution along axons (1, 9–11, 14), also observed in dendrites, but the extent to which it exists and how potentially affect a variety of signaling pathways in axons (1). The it develops in dendrites remain unclear. It is also unclear whether MPS can also act as a diffusion barrier and contribute to filtering other structural forms of the membrane skeleton are present in membrane proteins at the axon initial segment (21). In addition, the neurons. Here, we investigated the spatial organizations of spectrin, MPS appears to affect the stability of microtubules in axons (22). actin, and adducin, an actin-capping protein, in the dendrites and Subsequent to its observation in axons, the MPS structure was soma of cultured hippocampal neurons at different developmental also found to exist in the dendrites of wild-type neurons using stages, and compared results with those obtained in axons, using STORM and stimulated emission depletion microscopy (9, 10), superresolution imaging. We observed that the 1D MPS exists in a and its dendritic presence can be further enhanced by over- substantial fraction of dendritic regions in relatively mature neurons, expression of βII-spectrin in neurons (9). Although the MPS is but this structure develops slower and forms with a lower propensity generally observed throughout the entire axonal shaft of all main dendrites than in axons. In addition, we observed that spectrin, ture neurons (1, 9, 10, 12, 13), the extent to which this structure is actin, and adducin also form a 2D polygonal lattice structure, resem- present in dendrites is unclear and varies from study to study (9, bling the expanded erythrocyte membrane skeleton structure, in the 10, 13, 15, 16). In the two early studies reporting the 1D MPS in somatodendritic compartment. This 2D lattice structure also de- dendrites, a relatively small fraction of dendrites in wild-type velops substantially more slowly in the soma and dendrites than neurons were observed to exhibit this structure (9, 10), whereas the development of the 1D MPS in axons. These results suggest later studies reported either sparse or extensive presence of the membrane skeleton structures are differentially regulated across different subcompartments of neurons. 1D MPS in dendrites (13, 15, 16). It has been shown that the MPS develops early in axons, starting to appear as early as day 2 in vitro actin | spectrin | adducin | super-resolution imaging | STORM (DIV 2) (9, 10). The MPS first forms in the proximal axonal region near the soma and then propagates to the distal end of axons, t was recently discovered that actin, spectrin, and associated Significance Imolecules form a membrane-associated periodic skeleton (MPS) structure in neurons (1). As revealed by superresolution stochastic optical reconstruction microscopy (STORM) (1), this structure Actin, spectrin, and associated molecules form a quasi-1D periodic contains molecules homologous to those present in the erythrocyte membrane skeleton in neurons, which organizes membrane membrane skeleton, including spectrin (2–4), actin, and actin- proteins in periodic distributions and provides mechanical sta- capping proteins such as adducin (5, 6), but adopts a structural bility for axons. Here, we provide detailed quantifications of this organization that is distinct from the polygonal lattice structure of periodic structure in neurons and show that it develops substantially more slowly in dendrites than in axons. Moreover, we the erythrocyte membrane skeleton (7, 8). In this structure, short observed a 2D, polygonal lattice structure of these molecules in actin filaments capped by adducin are organized into repetitive, the somatodendritic compartment. The diverse structural orga- ring-like structures that wrap around the circumference of the axon nizations and different developmental courses of the membrane underneath the axonal membrane, and adjacent actin rings are skeleton in different neuronal compartments suggest the mem- connected by spectrin tetramers, forming a long-range quasi-1D ∼ brane skeleton is differentially regulated across these neuronal periodic structure with a periodicity of 190 nm (1). This periodic compartments. The observation of the polygonal lattice structure structure is formed extensively throughout the axonal shaft, in- in cells in addition to erythrocytes suggests a potentially general cluding the axon initial segment and the unmyelinated distal axons, presence of this structure across diverse cell types. as well as the nodes of Ranvier and internodal segments in mye- – linated axons (1, 9 14), but appears to be perturbed at synaptic Author contributions: B.H., R.Z., and X.Z. designed research; B.H., R.Z., and C.X. performed sites (13, 15, 16). It is a highly prevalent structure observed in many research; B.H. and R.Z. analyzed data; and B.H., R.Z., and X.Z. wrote the paper. different types of neurons, including both excitatory and inhibitory Reviewers: B.C., Stanford University; and V.M.F., The Scripps Research Institute. neurons, as well as neurons in both central and peripheral nervous The authors declare no conflict of interest. systems (12, 13), and is evolutionarily conserved across diverse Freely available online through the PNAS open access option. animal species, ranging from Caenorhabditis elegans and Drosophila 1B.H. and R.Z. contributed equally to this work. to rodents and humans (13). 2To whom correspondence may be addressed. Email: [email protected] or The MPS has been suggested to play a variety of functional [email protected]. roles in axons. Such a periodic skeletal structure formed by actin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rings connected by flexible spectrin tetramers could provide a 1073/pnas.1705043114/-/DCSupplemental.

E6678–E6685 | PNAS | Published online July 24, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1705043114 Downloaded by guest on September 24, 2021 spanning nearly the entire axonal shaft by DIV 7 (9). How the periodicity (Fig. 1 A–D). In comparison, we analyzed ∼200– PNAS PLUS MPS develops in dendrites is unclear. It also remains elusive 300 randomly selected axonal regions (also ∼3 μm in length) whether the molecular components of the MPS can organize into from the same DIV 28 neuron cultures and observed that nearly other structural forms in neurons. Here, we addressed these all axonal regions showed the 1D periodic pattern with ∼190-nm questions by investigating the distributions of βII-spectrin, βIII- periodicity (Fig. S1). Quantitatively, the average autocorrelation spectrin, adducin, and actin in the dendrites and soma at various amplitude derived from the dendritic regions was only ∼40% of developmental stages of cultured hippocampal neurons and that observed for the axonal regions, and the distributions of compared the results with those obtained in axons. autocorrelation amplitudes for dendritic regions were generally shifted to lower values compared with those for axonal regions Results (Fig. 1 E and F and Fig. S2). Spectrin, Actin, and Adducin Adopt 1D Periodic Distributions in a Next, we estimated the fractions of dendritic regions showing Substantial Fraction of Dendritic Regions in Relatively Mature the 1D MPS structures. Compared with the unbiased analysis of Cultured Neurons. We used 3D STORM imaging (23, 24) to ex- autocorrelation amplitudes, this fraction quantification has some amine the distributions of βII-spectrin, βIII-spectrin, actin, and ambiguity: Whereas some regions unambiguously exhibited either adducin in dendrites and axons of cultured mouse hippocampal a periodic or a nonperiodic distribution of these MPS molecules, neurons. Actin, adducin and βII-spectrin are abundantly expressed some regions are ambiguous in part because periodic and in both axons and dendrites (9, 25), whereas βIII-spectrin is a nonperiodic distributions coexist in (different portions of) β-spectrin isoform specifically enriched in dendrites with only a these regions (Fig. S2). Nonetheless, when the same criteria are low expression level in axons (26, 27). We used immunofluores- applied to such fraction quantification, results obtained from cence to label βII-spectrin, βIII-spectrin, and adducin, and dye- different neuronal compartments or under different conditions conjugated phalloidin to label actin. The antibody against βII- can be meaningfully compared. We adopted two methods to es- spectrin recognizes the C terminus of the protein, and hence the timate the fraction of regions showing the 1D MPS structure. In middle region of the spectrin tetramer; the antibody against βIII- the first method, we visually inspected the STORM images of spectrin recognizes the N terminus of the protein, and hence the these randomly selected ∼3-μm regions and classified regions as ends of the spectrin tetramer. We distinguished dendrites from exhibiting the 1D MPS structure if a ∼50% or greater portion of axons by the positive MAP2 immunoreactivity of dendrites. the region exhibited a periodic distribution. Using this approach, We observed 1D periodic distributions of actin, βII-spectrin, we found that ∼50–55% of the examined dendritic regions in DIV βIII-spectrin, and adducin in a substantial fraction of dendritic 28 neurons exhibited 1D periodic patterns for all four molecular regions in relatively mature mouse neurons at DIV 28. To markers: βII-spectrin, βIII-spectrin, actin, and adducin. In the quantify the degree of periodicity, we randomly selected ∼300– second method, we inspected the 1D autocorrelation functions 400 dendritic regions (∼3 μm in length) of DIV 28 neurons from and classified the regions showing more than two consecutive three independent biological replicates and performed 1D au- peaks at 190-nm intervals in the autocorrelation function as ex- tocorrelation analyses. For all four molecules, we observed that hibiting the 1D MPS structure. The fractions determined this way some of the dendritic regions exhibited 1D periodic distributions were similar (∼50–60% for the four molecular markers). In both with ∼190-nm intervals, whereas other regions exhibited little methods, some of the regions classified as being periodic contained

AB E Dendrite Actin DIV 28 βII spectrin Dendrite βII spectrin 0.8 DIV 28 βIII spectrin Dendrite βIII spectrin 0.8 MAP2 MAP2 0.8 DIV 28 Adducin n 0.0 0.0 βII spectrin lation βIII spectrin 1μm -0.8 -0.8 re 1μm 0 200 400 600 0 200 400 600

ocor 0.0 Lag (nm) Lag (nm) t Dendrite 0.8 Dendrite 0.8

Autocorrelation 0.0 Autocorrelatio 0.0 -0.8 0 200 400 600

-0.8 verage au -450 z 450 nm -0.8 0 200 400 600 0 200 400 600 A Lag (nm) Lag (nm) Lag (nm) C D F 0.8 0.8 Axon Actin DIV 28 Actin Dendrite Actin DIV 28 Adducin Dendrite Adducin n DIV 28 MAP2 MAP2 0.8 Adducin 0.0 0.0 βII spectrin relatio -0.8 -0.8 r 0 200 400 600 0 200 400 600

Lag (nm) toco 0.0 0.8 Lag (nm) 0.8 Dendrite Dendrite u Autocorrelation 0.0 Autocorrelation 0.0 -0.8 -0.8 -0.8 0 200 400 600 0 200 400 600 0 200 400 600 Lag (nm) Lag (nm) Average a Lag (nm)

Fig. 1. Spectrin, actin, and adducin adopt 1D periodic distributions in a substantial fraction of dendritic regions in relatively mature neurons. (A, Left) NEUROSCIENCE Conventional images of dendrites from DIV 28 mouse neurons stained for βII-spectrin (green) and a dendritic marker MAP2 (magenta). (Middle) 3D STORM images of βII-spectrin for the dendritic regions indicated by the yellow boxes on the Left and displaying 1D periodic (Top) and irregular (Bottom) spectrin distributions (the z-position information is color-coded). (Right) 1D autocorrelation functions of the white dashed boxed regions in the Middle.(B–D) Similar to A, but for DIV 28 neurons stained for βIII-spectrin (B), actin (C), and adducin (D) instead of βII-spectrin. We note that long actin filaments running along axons and dendrites could obscure the detection of periodic actin rings in the MPS. These long actin filaments are better preserved when the neurons arefixedby paraformaldehyde (PFA) plus glutaraldehyde (GA), but substantially diminished in neurons fixed by PFA alone, whereas the actin filaments in the MPS are well preserved under both fixation conditions. Thus, we used PFA fixation to minimize the obscuring effect of the longitudinal actin filaments on the detection of actin in the MPS (see SI Materials and Methods for more details). (Scale bars, 1 μm.) (E and F) Average autocorrelation functions calculated from ∼300–400 randomly selected dendritic regions in DIV 28 mouse neurons stained for βII-spectrin, βIII-spectrin, actin, and adducin (E), as well as from ∼200–300 randomly selected axonal regions in DIV 28 neurons stained for βII-spectrin, actin, and adducin (F), from three independent biological replicates for each condition.

Han et al. PNAS | Published online July 24, 2017 | E6679 Downloaded by guest on September 24, 2021 subportions that were nonperiodic, and some of the regions clas- 0.8 sified as being nonperiodic contained subportions that were peri- A odic. The fraction of regions exhibiting the 1D MPS structure and the average autocorrelation amplitudes of these molecular markers observed for spiny dendrites were similar to those observed on 0.6 βII spectrin Axon nonspiny dendrites. In comparison, we also performed the same Adducin Dendrite > fraction quantifications for axons and found that nearly all ( 95%) βIII spectrin Dendrite of the axonal regions were classified as exhibiting the 1D periodic βII spectrin Dendrite distribution for all three markers expressed in axons (βII-spec- 0.4 trin, actin, adducin), using both methods described earlier. The observed lengths of the periodic domains in dendrites Average varied from ∼1to∼30 μm, although overlap between neurites and the finite imaging field of view (40 × 40 μm) can truncate 0.2 periodic domains and cause an underestimate of the domain autocorrelation amplitude length. It is worth noting that, because a dendrite often contained both periodic and irregular regions, the fraction of den- 0.0 drites exhibiting at least one periodic region was quite large 7142128 48.5 (>80%), consistent with previous results (15, 16). We also observed heterogeneity among dendrites with some dendrites being Days in vitro (DIV) covered by the 1D MPS structure substantially more extensively than the others. Taken together, these results suggest nearly all B dendrites have some tendency to form the 1D MPS structure, but 1.0 the propensity of MPS formation and/or the regularity of the MPS structure within dendrites is lower than that within axons. βII spectrin Axon 0.8 Adducin Dendrite Developmental Course of the 1D MPS Structure in Dendrites. To in- βIII spectrin Dendrite vestigate the developmental course of the 1D MPS in dendrites, βII spectrin Dendrite we imaged the distributions of these MPS components in the 0.6 dendrites of cultured mouse hippocampal neurons at DIV 7, 14, 21, 28, 47, and 50 (Fig. 1; Figs. S3 and S4). As shown by the quantifications of the average 1D autocorrelation amplitudes and 0.4 the fractions of regions exhibiting the 1D periodic structure, the MPS appeared to develop substantially slower in dendrites than in axons (Fig. 2). We have shown previously that the 1D MPS starts Fraction of regions 0.2 to form in the proximal axon region near the soma at DIV 2 and then propagates to the distal region, spanning essentially the en- exhibiting 1D MPS structure tire axonal shaft by DIV 6–7 (9), as confirmed here (Fig. 2). The 0.0 observed increase in the autocorrelation amplitude after DIV 7 7142128 48.5 was primarily due to the increase in regularity of the 1D MPS structure in the periodic regions. In contrast, only a small fraction Days in vitro (DIV) of the dendritic regions showed the 1D MPS structure at DIV 7 Fig. 2. Developmental course of the 1D MPS structure in dendrites in com- (Fig. 2), and both the average autocorrelation amplitudes and the parison with axons. (A) The average autocorrelation amplitudes of βII-spectrin, fractions exhibiting 1D periodic pattern gradually increased during βIII-spectrin, and adducin observed for dendrites and the average autocorrela- the next several weeks (Fig. 2). In addition to mouse neurons, we tion amplitudes of βII-spectrin observed for axons of mouse neurons at DIV 7, 14, also examined cultured rat hippocampal neurons; although different 21, 28, 47, and 50. The autocorrelation amplitude is defined as the amplitude of species and culture conditions resulted in some quantitative differ- the first peak (at ∼190 nm) in the average autocorrelation function curve. ences, the slower development and lower formation propensity of (B) The fractions of dendritic and axonal regions classified as exhibiting the 1D the 1D MPS in dendrites, compared with axons, were also observed MPS structure at DIV 7, 14, 21, 28, 47, and 50 using the first method. In both A and B, we grouped data from DIV 47 and 50 into a single point, DIV 48.5. in the rat neurons (Fig. S5). Approximately 250–400 dendritic regions and ∼200–300 axonal regions from two to three independent biological replicates were analyzed for each molecular Effect of the Dendrite Diameter and Location on the Formation of the marker at each DIV. Error bars represent SEM. We also imaged actin at DIV 7 and 1D MPS Structure. The average diameter of dendrites (1.1 ± 0.5 μm) 28, and the fractions of dendritic regions exhibiting 1D periodic actin distribu- was greater than that of axons (0.52 ± 0.15 μm) in our cultured tions are similar to those observed for adducin, βII-spectrin, and βIII-spectrin. neuron system, raising the question whether the lower propensity of 1D MPS formation in dendrites observed here may stem from their relatively larger diameters. To test this, we analyzed den- membrane is nearly flat locally, and the membrane skeleton in such dritic regions with different diameters ranging from 0.4 to 2.6 μm wide neurites may adopt a different structural form. in DIV 28 mouse neurons. The 1D autocorrelation amplitudes We also examined whether the probability of 1D MPS for- showed little dependence on the dendrite diameter (Fig. 3 A–C), mation depends on the location of the dendritic region relative to suggesting the lower propensity for the 1D MPS formation in the soma. We found that the 1D autocorrelation amplitudes of dendrites we observed here was unlikely a result of their relatively these dendritic regions, as well as the fraction of dendritic regions larger diameters. However, it is worth noting that the range of classified as showing the 1D periodic structure, were independent diameters tested here was limited, and it is possible that over a of their relative distances to the soma (Fig. 3 D–F). This was true larger range of diameters, the formation of the 1D MPS may show both for the relatively mature neurons at DIV 28 (Fig. 3 D–F)and a dependence on the neurite diameter. In particular, the formation for neurons at an earlier developmental stage (DIV 7) when only a of the 1D MPS structure, which likely requires symmetry breaking, small fraction of the dendritic regions exhibited the 1D MPS may have a smaller tendency in very wide neurites in which the structure (Fig. S6 A–C).

E6680 | www.pnas.org/cgi/doi/10.1073/pnas.1705043114 Han et al. Downloaded by guest on September 24, 2021 PNAS PLUS A DIV 28 BCDIV 28 DIV 28 βII spectrin βIII spectrin Adducin r = -0.07 r = 0.16 r = -0.02 1.0 1.0 n on on o 1.0 tude itude relati i 0.5 relati 0.5 r r 0.5 orrelati co c co o amplitude ampl ampl t

0.0 u 0.0 Auto Aut o 0.0 A 0.00.51.01.52.02.5 0.00.51.01.52.02.5 0.00.51.01.52.02.5 Dendrite diameter (μm) Dendrite diameter (μm) Dendrite diameter (μm)

D DIV 28 E DIV 28 F DIV 28 1.5 βII spectrin r = 0.10 1.2 βII spectrin 0.8 βII spectrin βIII spectrin r = 0.15 βIII spectrin βIII spectrin 0.8 0.6 e 1.0 e rrelation d o ud u t 0.4 rrelation 0.4 plit o 0.5 utoc

ampli 0.2 am utoc 0.0 0.0 A exhibiting 1D MPS Fraction of regions 0.0

0.00.20.40.60.81.0 Average a 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative distance to soma Relative distance to soma Relative distance to soma

Fig. 3. Effect of the dendrite diameter and location on the 1D MPS formation. (A–C) Scatter plots showing the autocorrelation amplitudes versus the diameters of ∼100 randomly selected dendritic regions stained for βII-spectrin (A), βIII-spectrin (B), and adducin (C) for DIV 28 mouse neurons. The Pearson correlation coefficients (r) are indicated. (D) Scatter plots showing the autocorrelation amplitudes versus the relative distances to the soma for ∼100– 200 randomly selected dendritic regions stained for βII-spectrin (black) and βIII-spectrin (red) for DIV 28 mouse neurons. The relative distance is defined as the distance of the region to the soma along the dendrite divided by the full length of the dendrite containing this region. (E) The binned average autocorrelation amplitude as a function of the relative distance to the soma. Error bars represent SD. (F) Fraction of dendritic regions classified as exhibiting the 1D MPS structure as a function of the relative distance to the soma.

Role of βII-Spectrin in the 1D MPS Formation. Because βIII-spectrin (Fig. 4C). These results suggest βII-spectrin also plays a role in is the isoform of β-spectrin that is specifically enriched in den- the MPS formation in dendrite, consistent with the observation drites (26, 27), it is possible that βIII-spectrin is the major of βII-spectrin in the dendritic MPS. We note that although the β-spectrin component of the dendritic MPS structure, and that mRNA expression levels of βIII-spectrin and adducin were not βII-spectrin is not essential for the formation of this structure in changed by the knocking down of βII-spectrin (Fig. S7C), their dendrites. To test this possibility, we knocked down βII-spectrin protein levels were slightly reduced (Fig. S7D), potentially as the in DIV 28 mouse neurons, using a shRNA-expressing adenovirus result of a lower stability of these proteins in the absence of βII- (28). The mRNA and protein expression levels of βII-spectrin in spectrin. This reduction in the βIII-spectrin and adducin protein neurons were indeed greatly reduced (Fig. S7A), confirming the levels could also contribute to the disruption of the MPS in high knockdown efficiency. As expected, the MPS structure was dendrites by βII-spectrin knockdown. disrupted in axons (Fig. S7B) (9). Surprisingly, the MPS structure was also largely disrupted in dendrites after βII-spectrin knock- MPS Components Form 2D Polygonal Lattice Structures in the down, as evident from the STORM images of βIII-spectrin and Somatodendritic Compartment of Neurons. Interestingly, in some adducin in dendrites (Fig. 4). The average autocorrelation am- of the dendritic regions that did not exhibit a 1D periodic struc- plitudes for these molecules in dendrites were reduced to a level ture, the localization patterns of βIII-spectrin, adducin, and actin close to that obtained from neurons treated with actin depoly- did not appear entirely random; instead, the clusters of localiza- merizing drugs cytochalasin D (CytoD) and latrunculin A (LatA) tions appeared to exhibit a regular 2D lattice pattern. This reminded

A C Untreated DIV 28 βIII spectrin 0.8 Dendrite βIII spectrin on Control shRNA MAP2 ti LatA/CytoD

rela 0.0 βII spectrin shRNA r 0.6

1μm 1μm -0.8 *** *** *** βII spectrin shRNA *** *** *** Autoco 0 200 400 600 0.4 Lag (nm)

B 0.2 n DIV 28 Adducin Dendrite Adducin 0.8 Avergage io t

MAP2 a l

re 0.0 0.0 r n o i autocorrelationo amplitude c te

o duc 1μm 1μm t

d NEUROSCIENCE -0.8 spectrinndrite βII spectrin shRNA 0 200 400 600 I spectrin A Au II de dendrite dendri Lag (nm) β βII

Fig. 4. βII-spectrin dependence of the 1D MPS formation in dendrites. (A, Left) Conventional image of dendrites from βII-spectrin shRNA treated mouse neurons (DIV 28) stained for βIII-spectrin (green) and MAP2 (magenta). (Middle) 3D STORM image of βIII-spectrin for the dendritic region indicated by the yellow box on the Left.(Right) 1D autocorrelation function of the region indicated by the white dashed box in the Middle. (Scale bars, 1 μm.) (B) Similar to A, but for βII-spectrin knockdown neurons that were stained for adducin instead of βIII-spectrin. (C) The average autocorrelation amplitudes obtained for dendritic regions from untreated DIV 28 neurons and the DIV 28 neurons treated with control scrambled shRNA, βII-spectrin shRNA, and actin-depolymerizing drugs (50 μM CytoD and 20 μM LatA for 3 h), respectively. Because the MPS structure is relatively resistant to actin-depolymerizing drugs, a relatively high drug concentration and long treatment time is used to disrupt this structure. ∼100 dendritic regions were analyzed for each condition. Error bars represent SEM. ***P < 0.001 (unpaired Student’s t test).

Han et al. PNAS | Published online July 24, 2017 | E6681 Downloaded by guest on September 24, 2021 2D lattice Irregular 1D lattice DIV 28 βIII spectrin βIII spectrin βIII spectrin βIII spectrin A MAP2 ED

1μm 1μm 200 nm 200 nm 200 nm 200 nm DIV 28 Adducin Adducin Adducin Adducin B MAP2

DIV 28 Actin Actin Actin Actin C MAP2

F DIV 28 βIII spectrin G DIV 28 Adducin H βIII spectrin 2 MAP2 1 MAP2

2 200 nm 1 5μm 5 μm F1 F2 G1 G2 200 nm Adducin

200 nm 200 nm

IJK

βIII spectrin βIII spectrin βIII spectrin 0.8 Adducin 0.8 Adducin *** ** 0.6 0.6 ** ** 0.4 ** 0.4 ** ** 0.2 0.2

0.0 0.0 *

determined by connectivity DIV 7 DIV 7oD DIV 28 DIV 28 DIV 28 DIV 28 DIV 28 DIV 28 DIV28 DIV 28 DIV 28 DIV28 determined by 2D autocorrelation 200 nm /CytoD Fraction of regions exhibiting 2D lattice Fraction of regions exhibiting 2D lattice LatA/CytoD LatA/CytoD LatA/CytoD LatA LatA/Cyt spectrin shRNA spectrin shRNA Dendrite Soma βII βII Dendrite Soma

Fig. 5. MPS components form 2D polygonal lattice structures in some somatodendritic regions of neurons. (ALeft) Conventional image of a dendritic region from a DIV 28 mouse neuron stained for βIII-spectrin (green) and a dendritic marker MAP2 (magenta). (Middle Left) 3D STORM image of βIII-spectrin for the yellow boxed region on the Left.(Middle Right) Zoom-in image of the region indicated by the white dashed box in the Middle Left.(Right) 2D autocorrelation function of this boxed region, which shows a 2D periodic lattice pattern. [Scale bars, 1 μm (left two panels) and 200 nm (right two panels).] (B and C) Similar to A, but DIV 28 neurons were stained for adducin (B) and actin (C) instead of βIII-spectrin. (D) 2D autocorrelation functions of some dendritic regions exhibiting irregular distributions of βIII-spectrin (Top), adducin (Middle), and actin (Bottom). (E) 2D autocorrelation functions of some dendritic regions exhibiting 1D periodic distributions of βIII-spectrin (Top), adducin (Middle), and actin (Bottom). (Scale bars, 200 nm.) (F, Top) Conventional image of a DIV 28 mouse neuron stained for βIII-spectrin (green) and MAP2 (magenta). The image was obtained from the bottom (adhering) surface of the neuron to show the overall morphology of the neuron. (Middle and Bottom, F1 and F2) Two STORM images of βIII-spectrin for the orange arrow indicated regions in the top panel (Middle) and corresponding 2D autocorrelation functions of the regions marked by the white dashed boxes in the middle panels (Bottom). F1 contains regions that exhibit a 2D periodic lattice pattern. F2 exhibits an irregular distribution of βIII-spectrin. The STORM images were obtained from the top (dorsal) surface of the neuron. [Scale bars, 5 μm(Top) and 200 nm (Middle and Bottom).] (G) Similar to F, but for a DIV 28 neuron stained for adducin instead of βIII-spectrin. (H) STORM images of soma regions exhibiting the 1D periodic distribution of βIII-spectrin/adducin and the 2D autocorrelation functions of the regions marked by the white dashed boxes. (Scale bars, 200 nm.) (I) Fractions of the dendritic and soma regions containing the 2D lattice structure determined by the 2D autocorrelation analysis for untreated and LatA/CytoD-treated neurons. (J) STORM image of βIII-spectrin of a soma region (Top) and its reconstituted lattice network, using the method described in the main text (Top and Bottom). (K) Fractions of the dendritic and soma regions containing 2D polygonal lattice structures determined by the lattice reconstitution and connectivity method for untreated neurons, LatA/CytoD-treated neurons, and βII-spectrin knockdown neurons. *A value too close to zero to be visible in the bar graph. The fraction of dendritic regions exhibiting the 2D lattice structure is defined as the fraction among the regions that do not exhibit the 1D periodic structure (i.e., number of dendritic regions exhibiting the 2D polygonal lattice structure/number of dendritic regions not exhibiting the 1D periodic structure). Approximately 500–600 such dendritic regions not exhibiting the 1D periodic structure and ∼100– 200 soma regions were analyzed for each condition. Error bars represent SEM. **P < 0.05; ***P < 0.001 (unpaired Student’s t test).

E6682 | www.pnas.org/cgi/doi/10.1073/pnas.1705043114 Han et al. Downloaded by guest on September 24, 2021 us of the 2D polygonal lattice structure observed from the expanded regions had N values significantly beyond the values obtained PNAS PLUS membrane skeleton of erythrocytes (7, 8). If such a lattice structure is from simulations of random distributions (Fig. S10C). We then present in neurons, our labeling strategies for βIII-spectrin, adducin, determined a threshold value of N from the simulated distribu- and actin should mark the nodes of this polygonal lattice structure. tions and classified the experimentally imaged regions with N We used 2D autocorrelation analysis to probe the 2D periodicity in above this threshold as containing the 2D lattice structure (Fig. the organizations of these MPS components in the dendritic regions S10C). Using this approach, the fractions of regions classified as of DIV 28 neurons. Indeed, a small fraction (∼10%) of the regions containing the 2D lattice structure were calculated to be ∼40– that did not exhibit the 1D periodic pattern showed a 2D lattice 45% for the dorsal surface of the soma at DIV 28 from the patternwithtwoorthreeaxesof∼190-nm periodicity (Fig. 5 A–C), STORM images of βIII-spectrin and adducin, and these fractions which is distinct from the irregular patterns observed for other re- were substantially larger than the fractions derived from various gions (Fig. 5D). In contrast, the 2D autocorrelation analysis de- simulated random distributions (Fig. S10D). The fractions ob- tected only one axis of periodicity for those dendritic regions served for dendrites were smaller than those observed for soma that show the 1D periodic patterns (Fig. 5E). (Fig. 5K and Fig. S6D), but still larger than those derived from Because βIII-spectrin and adducin also showed an enrichment the simulated random distributions (Fig. S10E). The fractions at the plasma membrane of the soma (Fig. S8), we asked whether observed for both soma and dendrites were substantially reduced the 2D polygonal lattice structure could also be observed at the after LatA/CytoD treatment (Fig. 5K and Fig. S10F). Likewise, soma membrane. To answer this question, we randomly selected treatment with βII-spectrin shRNA also led to a drastic re- ∼100–200 regions (∼1 × 1 μm) on the top (dorsal) soma surface duction in the fractions of regions classified as exhibiting the 2D of DIV 28 mouse neurons and performed 2D autocorrelation lattice structure (Fig. 5K and Fig. S10F). In addition, we quan- analysis for βIII-spectrin or adducin distributions. A significant tified the densities of localization clusters for βIII-spectrin and fraction (∼20%) of these soma regions showed a 2D periodic adducin in the soma regions and found that the cluster densities lattice pattern with two or three axes of ∼190-nm periodicity decreased by ∼50–60% upon LatA/CytoD or βII-spectrin shRNA (average ∼187 nm) in the autocorrelation function (Fig. 5 F and treatment, which is consistent with the notion that the 2D lattice G). Some soma regions also exhibited the 1D periodic structure structure was disrupted by these treatments. (Fig. 5H). The ventral surface of the soma generally exhibited Taken together, these results suggest a membrane skeleton less βIII-spectrin and adducin staining, potentially as a result of with a 2D polygonal lattice structure is formed in the somato- adherence of the cell membrane to the glass substrate, and were dendritic compartment of neurons. It is possible that the 2D not further examined. lattice structure covers the somatodendritic surface more ex- Because of the finite areas of the regions examined, random tensively than what the fraction numbers indicate here because distributions of molecules could occasionally appear periodic by lattice deformation or incomplete labeling could obscure the chance in such finite-sized regions. The following two controls detection of the 2D lattice structure. suggest the 2D periodic patterns that we observed in the somato- Next, we investigated the development of 2D polygonal lattice dendritic compartment were not by random chance but reflected structures in the soma and dendrites. We used the lattice re- the tendency to form a 2D polygonal lattice-like structure: first, constitution and connectivity analysis as described earlier to simulated, randomly distributed clusters with the cluster density determine the fractions of regions in the soma and dendrites identical to the experimentally observed values showed a sub- containing the 2D polygonal lattice structures for DIV 7, 14, 21, stantially smaller probability to adopt such a 2D periodic pattern in and 28 mouse neurons. The fractions of regions classified as the 2D autocorrelation function (Fig. S9), and second, treatment containing the 2D lattice structure were small at DIV 7, and then of neurons with actin-depolymerizing drugs (CytoD and LatA) increased gradually as the neuron matures (Fig. 6), and the de- significantly reduced the fraction of regions exhibiting a 2D peri- velopmental course was similar to that of the 1D MPS in den- odic pattern in the 2D autocorrelation (Fig. 5I). drites, but substantially slower than that of the 1D MPS in axons. Because local lattice deformation or missing molecules in the lattice resulting from incomplete labeling could obscure the pe- Discussion riodic patterns in the 2D autocorrelation functions, such analyses We recently discovered that actin, spectrin, and associated might lead to an underestimate of the fraction of regions molecules form a 1D periodic membrane skeleton structure in showing the 2D lattice structure. Hence, we introduced an al- the axons of neurons (1). This 1D MPS structure is ubiquitously ternative quantification method by reconstituting the 2D lattice present in the axons of all of the neuronal types that have been network directly from the STORM images (Fig. 5J and Fig. examined so far (12, 13), and is conserved across diverse animal S10A). To this end, we identified clusters of localizations in each of the ∼1 × 1 μm soma regions, using a Voronoi tessellation based algorithm (29), and connected adjacent clusters with a line if their distance was close to the 190-nm periodicity of the lattice A 0.6 B 0.6 βIII spectrin Soma βIII spectrin Dendrite network derived from the autocorrelation analysis (see SI Ma- 0.5 Adducin 0.5 Adducin terials and Methods for details). The reconstructed lattice net- 0.4 0.4 work typically did not cover the entire area of the regions 0.3 0.3 examined (Fig. 5J), which could be a result of incomplete la- 0.2 beling and/or lattice distortion, but it is also possible that the 0.2 0.1 0.1 Fraction of regions

network was indeed not continuous everywhere on the cell sur- Fraction of regions exhibiting 2D lattice exhibiting 2D lattice 0.0 0.0 face. Next, to quantify the network for each region, we defined a 7142128 7 142128 NEUROSCIENCE connectivity number (N) as the number of nonoverlapping tri- DIV DIV angles observed in the reconstituted network per unit area of 1 μm2. Indeed, the average N value of the soma regions that Fig. 6. Developmental course of the 2D polygonal lattice structure in den- exhibited a pronounced 2D periodic pattern in the 2D autocor- drites and soma. (A and B) Fractions of the soma (A) and dendritic (B)regions exhibiting the 2D polygonal lattice structure determined by the lattice re- relation analysis was substantially larger than the N values constitution and connectivity method for DIV 7, 14, 21, and 28 mouse neurons. obtained from simulated, randomly distributed clusters with the The fraction of dendritic regions is defined as the fraction among the regions same density over the same area (Fig. S10B), and the scatterplot that do not exhibit the 1D periodic structure, as described in Fig. 5. Approxi- of N as a function of the cluster density for all the randomly mately 500–600 such dendritic regions and ∼100–200 soma regions were an- selected soma regions showed that a substantial fraction of these alyzed for each condition. Error bars represent SEM.

Han et al. PNAS | Published online July 24, 2017 | E6683 Downloaded by guest on September 24, 2021 species ranging from C. elegans to human (13). We and others This 2D MPS structure in neurons resembles the structural or- have also observed the 1D MPS in dendrites (9, 10, 13, 15, 16). ganization observed for the expanded membrane skeleton de- Here, we systematically investigated the distributions of four rived from erythrocytes, but the membrane skeleton observed in MPS components, βII-spectrin, βIII-spectrin, actin and adducin, intact erythrocytes appears much more rugged (7, 8). In fact, the in cultured hippocampal neurons. We observed that >80% of 190-nm periodicity we observed in neurons is close to the full dendrites in relatively mature neurons contain at least some length of the spectrin tetramer (7, 8), suggesting the possibility regions exhibiting 1D periodic distributions of these molecules, that the neuronal surface is under tension. Our observation of but regions with periodic and nonperiodic distributions often the polygonal lattice-like membrane skeleton structure in cells in coexist within the same dendrites. Overall, ∼50–60% of the addition to erythrocytes suggests this structure may be present in dendrite regions in relatively mature cultured neurons exhibit the a variety of cell types. 1D MPS structure. In comparison, more than 95% of axonal re- Similar to the 1D MPS structure, which has been shown to gions (essentially the entire axonal shaft) are covered by such 1D organize some functional membrane proteins into 1D periodic MPS structures under the same condition. In addition, the MPS distributions along the axons (1, 10, 14), the 2D MPS structure components tend to adopt highly ordered periodic distributions in observed here may also organize membrane proteins into 2D axons, whereas the spatial distributions of these molecules in periodic distributions on the surface of soma and neurites. It is dendrites often appear less regular, even for many of the dendritic worth noting that some ion channels and adhesion molecules at regions that are classified as exhibiting the 1D periodic structure. the nodes of Ranvier have recently been shown to adopt 2D The average amplitudes of the 1D periodic autocorrelation periodic distributions (14); however, these distributions do not functions in dendrites are substantially lower than those in axons. seem to arise from an underlying 2D periodic membrane skeleton Our results also showed that the 1D MPS develops sub- but, rather, appear to be imposed by the hexagonal organization of stantially slower in dendrites than in axons in the cultured hip- the microvilli of glial cells, as the axonal membrane skeleton at the pocampal neuron system. This structure starts to appear in axons nodes of Ranvier still adopts a 1D periodic structure (14). as early as DIV 2, and by DIV 7, essentially the entire axonal Our quantifications show that the 2D polygonal lattice struc- shaft is covered by the 1D MPS structure (9, 10). In contrast, the ture exists in a larger fraction of regions in the soma than in 1D MPS structure begins to appear in dendrites substantially dendrites. This is possibly because the 2D polygonal lattice later than DIV 2, and the coverage of the structure in dendrites preferentially forms at relatively flat surfaces, whereas the 1D gradually grows over several weeks. Because our study is based MPS structure preferentially forms at tubular surfaces with rel- on imaging of fixed neurons, our results do not exclude the atively large curvatures. Hence, it is conceivable that the 2D possibility that a more labile form of the 1D MPS is formed lattice structure may have a higher tendency to form in dendrites, earlier or more quickly, but the stable structural form of the MPS and possibly axons as well, that are substantially wider than those that can survive the fixation develops over this slow developmental studied here. Similar to the 1D MPS structure in dendrites, we course. This slow course could partially explain the varying extent observed that the development of the 2D MPS structure in the to which the 1D MPS has been observed in dendrites in previous somatodendritic compartment is also substantially slower than reports. Indeed, in the studies that examined relatively young wild- the development of the 1D MPS in axons. type neurons, a small fraction of the dendritic regions was observed The diverse structural organizations and the different devel- to exhibit the 1D MPS structure (9, 10, 13), whereas in the studies opmental courses of the membrane skeleton structures observed that imaged relatively mature neurons, a more extensive presence in the different subcompartments of neurons suggest the actin- of the 1D MPS was observed (15, 16). spectrin–based membrane skeleton is differentially regulated Taken together, our results suggest that nearly all dendrites across these neuronal subcompartments. The functional im- have some tendency to form the 1D MPS structure, but the plications of these differences in structures and dynamics await propensity of formation and rate of development of this struc- further investigation. ture are lower in dendrites than in axons. Whether the dendrite– axon differences observed here for the cultured hippocampal Materials and Methods neurons extend to other neuronal types remains to be de- Experimental procedures for neuronal culture; immunofluorescence labeling termined. We also note that cultured neurons, even at their late of βII-spectrin, βIII-spectrin, and adducin; and phalloidin labeling of actin, developmental stage with spines and synapses formed, may still drug and shRNA treatments, STORM imaging, and image analyses are de- not be as mature in some aspects as neurons in adult animals. scribed in the SI Materials and Methods. Therefore, it remains an open question as to what extent the 1D MPS exists and how fast it develops in the dendrites in vivo. ACKNOWLEDGMENTS. We thank Dr. Matthew Rasband (Baylor College of Medicine) for providing the adenoviruses expressing shRNA against βII-spec- Notably, in addition to the 1D MPS structure, we also ob- trin. This work is supported in part by the NIH. R.Z. is a Howard Hughes served a 2D polygonal lattice structure formed by the MPS Medical Institute Fellow of the Life Sciences Research Foundation. X.Z. is a components in the somatodendritic compartment of neurons. Howard Hughes Medical Institute investigator.

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